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Conjugation of tetracycline and penicillin with Sb(v) and Ag(i) against breast cancer cells

  • Paraskevi Z. Trialoni , Zografia-Christina M. Fyrigou , Christina N. Banti EMAIL logo and Sotiris K. Hadjikakou EMAIL logo
Published/Copyright: September 7, 2022
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Main Group Metal Chemistry
From the journal Main Group Metal Chemistry Volume 45 Issue 1

Abstract

Tetracycline (TecH 2 ) reacts with triphenylantimony (TPSb iii ) in the presence of hydrogen peroxide to form the [Ph3Sbv(Tec)] (TecAn). The sodium penicillin G (PenH) conjugates with Ag(i) towards [Ag(Pen)(MeCN)]2 (PenAcAg). TecAn and PenAcAg were characterized by melting point, X-ray fluorescence spectroscopy, attenuated total reflectance-Fourier transform infra-red, thermogravimetric-differential thermal analysis in solid state, ultraviolet-Vis spectroscopy, and nuclear magnetic resonance (1H and 13C-NMR), spectroscopies in solution. The molecular weight was determined with cryoscopy. The in vitro cytotoxic activity of TecAn and PenAcAg was evaluated against the human breast adenocarcinoma cell lines: MCF-7 (positive to hormones receptor (HR+)), MDA-MB-231 (negative to hormones receptor (HR−)), and their in vitro toxicity and genotoxicity were tested against normal human fetal lung fibroblast cells (MRC-5). The MCF-7 cells’ morphology and acridine orange/ethidium bromide staining suggest an apoptotic pathway for cell death. The binding affinity of TecAn and PenAcAg with DNA was, ex vivo, studied by UV-Vis and fluorescence spectroscopy and viscosity measurements of DNA solution. PenAcAg inhibits lipoxygenase (LOX) stronger than cisplatin, while no inhibitory activity has been detected for TecAn. The reduction of non-active Sb(v), of TecAn, to active Sb(iii) by glutathione (a tripeptide over expressed in tumor cells) was also investigated.

Graphical abstract

Keywords: biological inorganic chemistry; organoantimony(V); silver(I); antibiotic; cytotoxicity; DNA–metal complexes interaction

1 Introduction

Although treatments have been evolved, breast cancer remains the most common malignancy in women (Llewellyna et al., 2019). Depending on the breast cancer cells’ response to hormones, the disease is classified as hormone-dependent, when there is over expression of estrogen or progesterone receptors, or hormone-independent when the progression of the disease is independent to the hormone levels (The Cancer Genome Atlas Network, 2012).

Antibiotics promote cancer cell apoptosis, inhibit the growth, and prevent their metastasis (Gao et al., 2020). Among antibiotics, the group of anthracyclines have been studied for their antiproliferative activity (Gao et al., 2020). Doxorubicin, an antibiotic of anthracyclines family, has a broad anticancer spectrum (Gao et al., 2020). It is widely used to treat various types of cancer, including breast cancer (Christowitz et al., 2019). Its mechanism involves breakage of DNA strands, since DNA is one of the major molecular targets of antibiotics (Gao et al., 2020; Rocha et al., 2018). Besides, tetracycline exhibits structural similarity to doxorubicin (Fuoco, 2012; Mealey et al., 2002). N-Methylthio β-lactams, on the other hand, is a class of drugs that have been found to induce apoptosis in several cancer cell lines, including breast, prostate, head, and neck cancers, and leukemia (Bhattacharya and Mukherjee, 2015). Penicillin is the most common beta-lactam antibiotic (Macy, 2014). Therefore, it is of interest to investigate whether an antiproliferative activity of conjugates of tetracycline or penicillin with main group metals and/or metals is exhibited as well.

Antimonials are used for the treatment of fever, pneumonia, inflammatory diseases, and leishmaniasis (Ozturk et al., 2014; Polychronis et al., 2019). Although, Sb(iii) compounds are more active than those of Sb(v), the latter are used as pro-drugs due to their lower toxicity. Once these compounds enter into cytoplasm, Sb(v) is reduced to Sb(iii) due to the specific microenvironmental conditions of cancer cells, like the low O2 level or the presence of the tripeptide glutathione (GSH). GSH is over expressed in the cancerous cells participating in the cell resistance mechanism (Polychronis et al., 2019). Silver(i) compounds, on the other hand, interact with nuclear DNA and lipoxygenase (LOX), causing cells’ apoptosis through the mitochondrial signaling pathway (Banti et al., 2016). Recently, the conjugate of penicillin G (PenH) with silver(i) ions with formula [Ag(pen)(MeOH)]2 (PenAg) was synthesized, characterized, and studied for its antibacterial activity against Gram negative and positive bacterial strains (Ketikidis et al., 2020).

In the course of our studies in the field of drug design and development of new chemotherapeutics for breast cancer treatment (Banti et al., 2015, 2016, 2021; Chrysouli et al., 2018; Ketikidis et al., 2020; Latsis et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021; Tsiatouras et al., 2016), from the conjugation of antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) with metal ions, here we report the conjugation of TecH2 and PenH (Scheme 1), with antimony(v) and silver(i), respectively, with formulae [Ph3Sb(Tec)] (TecAn) and [Ag(Pen)(MeCN)]2 (PenAcAg). The new compounds were characterized by melting point, X-ray fluorescence spectroscopy (XRF), attenuated total reflectance-Fourier transform infra-red (ATR-FTIR), thermogravimetric-differential thermal analysis (TG-DTA), ultraviolet-visible (UV-Vis) spectroscopy, nuclear magnetic resonance (1H and 13C-NMR). PenAcAg is also obtained by the already known [Ag(Pen)(MeOH)]2 (PenAg) by its treatment with acetonitrile (Ketikidis et al., 2020). The in vitro cytotoxicity of TecAn and PenAcAg was evaluated towards MCF-7 and MDA-MB-231 cell lines. The in vitro toxicity and genotoxicity of both compounds were also tested against MRC-5 cell line. The apoptotic type of MCF-7 cell death was confirmed by cell morphology and acridine orange/ethidium bromide (AO/EB) staining. The ex vivo mechanism of TecAn and PenAcAg was clarified by CT-DNA interaction studies and LOX studies. In the case of TecAn, its reaction with GSH was also evaluated.

Scheme 1 Molecular diagram of TecH2 and PenH.
Scheme 1

Molecular diagram of TecH2 and PenH.

2 Results and discussion

2.1 General aspects

TecAn was synthesized by reacting TecH2 with Ph3Sb in the presence of hydrogen peroxide (Scheme 2). The pale-yellow precipitation was filtered off and dried in room temperature. PenAcAg was obtained by reacting equimolar amount of silver nitrate (AgNO3) with the sodium salt of penicillin G (PenNa) (1:1) in double distilled water (ddw) (Scheme 2) (Ketikidis et al., 2020). The powder products are purified by dissolving them in acetonitrile following by centrifugation at 2,000 rpm. The clear supernatant solutions were concentrated in the rotary evaporator to dryness and the yellow solids TecAn and PenAcAg were collected with diethylether (Et2O). Several attempts have been made to grow crystals using various solvents and crystallization methods without success. TecAn and PenAcAg are air stable when are stored in darkness at room temperature. TecAn is highly soluble in CH2Cl2, CHCl3, DMF, Dimethylsulfoxide (DMSO), and acetone and soluble in MeOH and MeCN, while PenAcAg is highly soluble in CH2Cl2, CHCl3, MeCN, DMF, and DMSO and soluble in MeOH.

Scheme 2 Reaction route for the synthesis of TecAn (a) and PenAcAg (b).
Scheme 2

Reaction route for the synthesis of TecAn (a) and PenAcAg (b).

2.2 XRF spectroscopy

The XRF spectrum of TecAn powder confirms the presence of Sb in the complex (Figure 1). The content of Sb in TecAn was determined at 14.86 (±0.09)% w/w (calc. for [Ph3Sb(Tec)] 15.31% w/w). The content of Ag in the case of PenAcAg was found 19.81 (±0.27)% w/w (calc. for [Ag(Pen)(MeCN)]2 22.4% w/w).

Figure 1 XRF spectra of TecAn (a) and PenAcAg (b).
Figure 1

XRF spectra of TecAn (a) and PenAcAg (b).

2.3 Cryoscopic molecular weight (MW) measurements

The MWs of the compounds were measured with cryoscopy in DMSO/ddw (1:49 v/v) solution using freezing point osmometer. A solution of 1 μL TecAn or PenAcAg (1 mg/100 μL DMSO) was diluted to 50 μL ddw. The MW was found to be 799 g·mol−1 for TecAn (calc. 795.5 g·mol−1 for [Ph3Sb(Tec)]) and 912 g·mol−1 for PenAcAg (calc. 946 g·mol−1 for [Ag(Pen)MeCN]2).

2.4 ATR-FTIR

The ν(H–Caromatic) and ν(H–Caliphatic) bands in the IR spectrum of TecH 2 at 3,050 and 2,958 cm−1, respectively, (Leypold et al., 2003) are observed in the spectrum of TecAn (Figures S1–S3 in Supplementary material). The vibrational band at 1,640 cm−1 in the FTIR spectrum of tetracycline is assigned to the ν(amid-CO) and ν(O═C(3)) vibration bands (Scheme 1) (Leypold et al., 2003). This band is shifted by 12 cm−1 (at 1,652 cm−1) in the spectrum of TecAn implying coordination of TecH 2 to Sb(v) through either the amide O(C1) or the carbonyl O(C3) donor atom. The vibrational bands at 1,328 and 1,302 cm−1 in the IR spectrum of TecH 2 are attributed to the O–C(13) and H–OC(13) bond vibrations (Scheme 1) (Leypold et al., 2003). These bands are observed at 1,335 and 1,302 cm−1 in the spectrum of TecAn indicating the non-involvement of the O[C(13)] atom in the coordination to Sb. The vibrational band at 410 cm−1 in the IR spectrum of TecAn is assigned to the Sb–O bond vibration (Bordner et al., 1986). Therefore, a coordination of TecH2 to Sb(v) through amide C(1) and carbonyl C(3) oxygen atoms is concluded by ATR-FTIR spectroscopy.

The assignment of the FTIR spectrum of PenAcAg was based on the already reported one (Anacona and Figueroa, 1999; Ketikidis et al., 2020). Briefly, the v as(–COO–) and v s(–COO–) of the carboxylic group in the IR spectrum of PenNa at 1,619 and at 1,416 cm−1 (Figure S4) are shifted in the case of PenAcAg at 1,576 and 1,394 cm−1, respectively (Figure S5) (Anacona and Figueroa, 1999; Ketikidis et al., 2020). The Δv [v as(COO−)–v s(COO−)] difference value of the ionic salt of penicillin PenNa is 203 cm−1, while the corresponding value Δv for PenAcAg is 182 cm−1 supporting the coordination of the ligand to the metal center through the carboxylic acid group. Taking into consideration that the Δv of PenAcAg is close to the corresponding one of PenNa, a bridging coordination mode of the carboxylic group of penicillin G to Ag(i) is concluded. This is in accordance with the structure already proposed for [Ag(Pen)MeOH]2 (Anacona and Figueroa, 1999; Ketikidis et al., 2020). The vibration band of the keto-group (C═O) of β-lactam at 1,774 cm−1 in PenNa remains unchanged in PenAcAg (at 1,767 cm−1) suggesting no involvement of this group in the coordination of PenNa into Ag(i). Thus, PenNa coordinating to Ag(i) through carboxylic group in a bridging mode is concluded by ATR-FTIR spectroscopy.

2.5 TG-DT analysis

TG/DTA analysis was performed under air flow with a rate of 10°C·min−1 up to 500°C. Two main decompose endothermic steps are observed in the case of TecAn: the first one occurs at 170–200°C with 10.3% mass loss which corresponds to the evolution of one phenyl group (Ph-H) from TPSb (calc. 9.8%) and the second one at 200–285°C with mass loss 19.3% which corresponds to the evolution of two phenyl groups (Ph-H) of TPSb (calc. 19.6%) (Figure S6). The TG/DTA thermograph shows that PenAcAg decomposes in two steps: the first is observed at 70–140°C with 6.7% mass loss which corresponds to the evolution of one acetonitrile (calc. 8.5%) and the second occurs at 150–140°C with 57.3% which corresponds to the evolution of one PenH (calc. 69%) (Figure S7).

2.6 UV-Vis spectroscopy

The UV-Vis absorption spectra of TecAn, TPSb, and TecH 2 were recorded in DMSO solution (Figure S8). Two absorption bands at 368 nm (ε = 17,104 cm−1·M−1) and 266 nm (ε = 25,080 cm−1·M−1) are observed in the spectrum of TecH 2 , which are assigned to π* ← π transitions. The spectrum of TPSb is dominated by one absorption band at 266 nm (ε = 10,740 cm−1·M−1) which is attributed to π* ← π electrons excitation. The corresponding spectrum of TecAn shows three absorption bands at 378 nm (ε = 16,270 cm−1·M−1), at 304 nm (ε = 13,964 cm−1·M−1) and at 266 nm (ε = 13,116 cm−1·M−1). The new absorption band at 304 nm (ε = 13,968 cm−1·M−1) in the spectrum of TecAn is assigned to metal to ligand charge transfer transition. The bands at 378 and 266 nm are assigned to intraligand π* ← π transitions. The corresponding UV-Vis spectra of PenAcAg and PenH in DMSO solution show one transition band at λ max 269 nm (ε = 5,795 cm−1·M−1) which is assigned to intraligand π* ← π transitions and 262 nm (ε = 1,310 cm−1·M−1), respectively (Figure S9)

2.7 Stability studies

The stability of TecAn and PenAcAg in DMSO-d 6 solution was tested by 1H-NMR spectroscopy (Figures S10 and S11) for 48 h. No changes were observed between the initial 1H-NMR spectra and the corresponding ones recorded after 48 h, confirming the retention of the structures of both TecAn and PenAcAg in solution.

2.8 1H-NMR studies

2.8.1 TecAn

The 1H-NMR spectra of TecH2, TPSb, and TecAn in DMSO-d 6 are shown in Figure 2 and Figure S12. The broad resonance signals at 9.15 and 8.78 ppm in the spectrum of TecH2 are attributed to the amide H[NH2–C(1)═O] (Scheme 1) protons (Williamson and Everett, 1975). These signals are observed at 9.21 and 9.25 ppm, respectively, in the spectrum of TecAn. The resonance signals at 7.54 ppm in the spectrum of TecH2 is assigned to the H[C(8)aromatic] of D ring (Scheme 1), at 7.12 ppm is attributed to the H[C(9)aromatic] (Scheme 1), and the signal at 6.91 ppm corresponds to the H[C(7)aromatic] (Scheme 1) (Williamson and Everett, 1975). Upon coordination of TecH2 to Sb(v) ion in TecAn, these signals are shifted upfield at 7.37, 7.00, and 6.77 ppm respectively, suggesting deprotonation of the hydroxyl H[O–C(6)] group. The aromatic protons of the phenyl groups of TPSb appear at 7.39–7.31 ppm. These signals are observed at 7.74–7.62 (o-H[Ph-Sb]) and 7.44–7.42 ppm (p-H[Ph-Sb]) in the spectrum of TecAn. The broad signal at 5.06 ppm in the spectrum of TecH2 corresponds to the hydroxyl H[O–C(10)] proton (Williamson and Everett, 1975), which is observed at 4.88 ppm in the spectrum of TecAn. The resonance signal at 2.87 ppm in the spectrum of TecH2 is assigned to H[C(10a)] proton, while the signal at 2.0 ppm is assigned to the H[C(11)] proton (Scheme 2). These signals are observed at 2.87 and at 2.04 ppm, respectively, in the spectrum of TecAn. The signal at 2.41 ppm corresponds to the H[H3C(15)] and H[H3C(16)] of TecH2, while it appears at 2.61 ppm in TecAn. The signal at 1.50 ppm is attributed to the methyl protons of H[H3C(14)] and it remains unchanged. Assignment of the 1H-NMR spectrum of TecAn results in the Δδ values with respect to the corresponding ones in TecH2 in DMSO-d 6 (Figure 3). The protons that strongly shifted are H[NH–C(1)═O], H[H3C(15)] and H[H3C(16)] due to the coordination of TecH2 to Sb through amide and carbonyl oxygen atoms (Scheme 1). The integration of the o-H[Ph-] of phenyl substituent of TPSb in TecAn (6 protons) with those of methylene H[C(14)] (3 protons) confirms the 1:1 molar ratio of Sb:TecH2 in TecAn (Figure 2).

Figure 2 1H-NMR spectra of TecAn in DMSO-d 6.
Figure 2

1H-NMR spectra of TecAn in DMSO-d 6.

Figure 3 The Δδ values in 1H-NMR of TecAn and PenAcAg with respect to the corresponding ones in TecH2 and PenNa in DMSO-d 6.
Figure 3

The Δδ values in 1H-NMR of TecAn and PenAcAg with respect to the corresponding ones in TecH2 and PenNa in DMSO-d 6.

2.8.2 PenAcAg

The 1H-NMR spectra of PenNa and PenAcAg in DMSO‑d 6 are shown in Figure 4 and Figure S13. The resonance signal at 8.66 ppm in the spectrum of PenNa is attributed to the amide proton H[NH–C(6)═O] (Scheme 1) (Branch et al., 1987). This signal is shifted at 8.81 ppm in the spectrum of PenAcAg. The resonance signals at 7.29–7.17 ppm in the spectrum of PenNa are assigned to the aromatic protons of the benzyl group (Scheme 1) (Ben Salem et al., 2016). These signals are observed at 7.32–7.22 ppm in the case of PenAcAg. The signals at 5.26 ppm and 5.28 ppm in the spectrum of PenNa are assigned to H[C(9)] proton and H[C(8)] proton, respectively (Ben Salem et al., 2016). These signals are downfield shifted at 5.48 and at 5.40 ppm, respectively, in the spectrum of PenAcAg. The resonance signal at 3.78 ppm corresponds to the H[C(12)] proton of PenNa, while for PenAcAg it appears at 4.14 ppm. This strong shift (Δδ = 0.36) confirms the coordination of the carboxylic group with the Ag(i). The signal at 3.5 ppm is attributed to H[C5] proton (Figure 3) (Ben Salem et al., 2016) and remains unshifted in PenAcAg. The resonance signal to 2.09 ppm is assigned to H[CH3CN] of the coordinated acetonitrile to the Ag(i). The corresponding signal of free H[MeCN] is observed at 2.07 ppm (Gottlieb et al., 1997). The resonance signals at 1.53 and 1.41 ppm are attributed to H[C(16)] and H[C(17)] protons (Ben Salem et al., 2016), respectively, while in the spectrum of PenAcAg they appear at 1.60 and 1.48 ppm, respectively. The Δδ values between PenNa and PenAcAg in DMSO-d 6 are shown in Figure 3. The protons that strongly shifted are H[C(12)] H[C(9)] and H[C(8)] due to the coordination of PenH to Ag through carboxylic oxygen atoms Sb (Scheme 1). The integration of the H[C(16)] (3 protons) with those of H[MeCN] (3 protons) confirms the 1:1 molar ratio of Ag:PenH in PenAcAg (Figure 4).

Figure 4 1H-NMR spectra of PenAcAg in DMSO-d 6.
Figure 4

1H-NMR spectra of PenAcAg in DMSO-d 6.

2.9 Molecular structure elucidation

Based on the analytical and spectroscopic data from XRF, cryoscopy, FTIR, and 1H-NMR the following possible formulae (Scheme 3) can be concluded for TecAn and PenAcAg.

Scheme 3 A possible formulae of TecAn and PenAcAg with charge distribution.
Scheme 3

A possible formulae of TecAn and PenAcAg with charge distribution.

2.10 Biological studies

2.10.1 In vitro antiproliferative activity

The in vitro antiproliferative activity of TecAn and PenAcAg was tested against two human adenocarcinoma breast cell lines, MCF-7 (hormone depended (HD)) and MDA-MB-231 (hormone independent (HI)) by sulforhodamine B (SRB) assay after their incubation for 48 h. The IC50 values of TecAn and PenAcAg against MCF-7(HD) are 26.0 ± 1.4 and 8.1 ± 0.2 μM, respectively, while the corresponding values against MDA-MB-231(HI) are 27.8 ± 0.9 and 8.9 ± 0.2 μM, respectively (Table 1). The antibiotics TecH2 and PenNa were inactive against both cell lines in the concentrations tested (up to 40 μM) (Table 1). Therefore, the conjugation of tetracycline with TPSb and penicillin with Ag(i) enhances their antiproliferative activity. The IC50 values of cisplatin are 6.8 ± 0.3 μM (MCF-7(HD)) and 26.7 ± 1.1 μM (MDA-MB-231(HI)). Hence, PenAcAg reveals 3-folds stronger activity than cisplatin against MDA-MB-231(HI) malignant cells. Both agents exhibit similar activity against MCF-7 (HD) and MDA-MB-231(HI) cells suggesting that hormone receptors might not involve in their mechanism of action.

Table 1

Antiproliferative activity of compounds against MCF-7 (HD), MDA-MB 231 (HI), and MRC-5 cells

IC50 (μM) TPI K b (×104) M−1 K app (×104) M−1 LOX inhibition IC50 (μM) Ref.
MCF-7 MDA-MB 231 MRC-5 MCF-7 MDA-MB 231
TecAn 26.0 ± 1.4 27.8 ± 0.9 24.5 ± 1.0 0.94 0.88 11.1 ± 0.5 1.1 ± 0.2 >60 *
TecH 2 >40 >40 >40 7.2 ± 0.8 0.8 ± 0.4 >40 *
Ph 3 Sb >30 >30 >30 — 5.3 ± 0.5 ND (Banti et al., 2016)
PenAcAg 8.1 ± 0.2 8.9 ± 0.2 7.9 ± 0.2 0.98 0.89 15.6 ± 5.1 6.7 ± 1.1 19.8 *
PenNa >30 >30 — 0.9 ± 0.1 >40 *
[Ph 3 Sb(Carv) 2 ] 7.1 ± 0.2 7.2 ± 0.3 8.1 ± 0.3 1.1 1.1 — 3.10 ± 0.43 ND (Kapetana and Banti, 2022)
{[Ph 3 Sb(SalH)] 2 O} 11.9 ± 0.6 8.0 ± 0.4 7.8 ± 0.2 0.7 1.0 30 ± 5.0 ND NA (Polychronis et al., 2019)
Cisplatin 5.5 ± 0.4 26.7 ± 1.1 1.1 ± 0.2 0.20 0.04 65.9 (Banti et al., 2016)

  1. *This work; Carv = carvacrol; SalH2 = salicylic acid; NA = no activity; ND = not determined.

2.10.2 In vitro toxicity study

The toxicities of TecAn and PenAcAg were examined against normal human fetal lung fibroblast cells (MRC-5). Their IC50 values of TecAn and PenAcAg against MRC-5 cells are 24.5 ± 1.0 and 7.9 ± 0.2 μM, respectively, while the corresponding value of cisplatin is 1.1 ± 0.2 μM. The Therapeutic Potency Index (TPI), which is defined as the IC50 of an agent against non-cancerous cells toward its IC50 against cancerous cells, for TecAn and PenAcAg are 0.94 and 0.98 against MCF-7 (HD) and 0.88 and 0.89 against MDA-MB-231(HI), respectively (Table 1). Given that the corresponding TPI values of cisplatin against MCF-7 (HD) and MDA-MB-231(HI) are 0.20 and 0.04, TecAn and PenAcAg are significantly more effective against cancerous than normal cells from cisplatin. Moreover, US Food and Drug Administration (FDA) defines an agent with no selectivity when it exhibits minimum toxic concentration (MTC)/minimum effective concentration (MEC) value less than 2 (Abughazaleh and Tracy, 2014). Thus, both TecAn and PenAcAg should be considered as toxic agents as well as cisplatin which however is an anticancer drug with clinical use.

2.10.3 In vitro genotoxicity study

The in vitro genetic damage caused by TecAn and PenAcAg towards MRC-5 cells was evaluated by the micronucleus (MN) assay. MNs are formed during the metaphase anaphase transition of the mitosis of a normal cell under the influence of a xenobiotic agent (such as chemicals) (Banti et al., 2021). The MRC-5 cells were incubated by TecAn and PenAcAg at their IC50 value. The MN frequency of the untreated cells is 1.0 ± 0.3%. When the MRC-5 cells were treated with TecAn, the MN frequency is 1.6 ± 0.1%, showing similar genotoxicity with that of control, while the MN is 2.0 ± 0.4%, when PenAcAg is used. The genotoxicity caused by TecAn and PenAcAg is similar than that of cisplatin, (MN frequency 1.6%) (Banti et al., 2016) (Figure 5).

Figure 5 Snapshots of MN formed in untreated MRC-5 cells (a) and upon their treatment with TecAn (b) and PenAcAg (c) for 48 h.
Figure 5

Snapshots of MN formed in untreated MRC-5 cells (a) and upon their treatment with TecAn (b) and PenAcAg (c) for 48 h.

2.10.4 In vivo toxicity studies

In order to examine the in vivo toxicity of TecAn, the brine shrimp Artemia salina assay was used (Banti and Hadjikakou, 2021). This assay is selected because it can predict a variety of biological activities of an agent, such as cytotoxic, phototoxic, pesticidal, and pharmacological activities of bioactive compounds (Banti and Hadjikakou, 2021; Stathopoulou et al., 2021). Therefore, several characteristics of brine shrimp such as its widespread distribution, short life cycle, non-selective grazing, and sensitivity to toxic substances (Banti and Hadjikakou, 2021), make it the ideal candidate to conduct this test.

The survival rate (%) of Artemia salina larvae in increasing concentrations of solutions with or without TecAn and TecH2 after 24 h is evaluated and the lethality was noted in terms of deaths of larvae. The concentrations used were 25, 50, and 100 μM. For the compounds the survival rates are up to 100% at all concentrations although it rises even up to 4-fold higher than the IC50 values for TecAn. No mortality rate of brine shrimp larvae was found upon their incubation with PenAg in concentrations up to 220 μM (Ketikidis et al., 2020). This survival rate indicates no toxicity of TecAn and PenAg at their IC50 values and at higher concentrations (Ketikidis et al., 2020).

2.10.5 In vitro mechanism of action

The mechanism of action of TecAn and PenAcAg against MCF-7 (HD) cells was examined in vitro by the means of cell morphology studies and AO/EB Staining. Moreover, the molecular mechanism was further studied ex vivo by their binding affinity towards CT-DNA using UV-Vis, fluorescence spectroscopy, and viscosity studies. The inhibitory activity of TecAn and PenAcAg towards LOX, an enzyme which catalyzes the oxidation of linoleic acid to hydroperoxy linoleic acid during the inflammation process, is also studied ex vivo. The low active antimonials(v) pro-drugs, such as TecAn, on the other hand, can readily be converted to the active drugs Sb(iii) by GSH. GSH is a tripeptide that is over-expressed in tumor cells, and it is involved in the development of cancer cells’ resistance to chemotherapeutics drugs. The redox reaction between the GSH and TecAn is studied, here, using vibrational spectroscopy.

2.10.5.1 Cell morphology studies

MCF-7 cells are treated with TecAn and PenAcAg at their IC50 values for 48 h and they are observed in inverted microscope, in order to assess the type of their death by their morphology. Figure 6 shows the morphological changes. The proliferation of the untreated MCF-7 (HD) cells is normal since they are elongated, adherent, and showed cellular crowding. However, the morphology of those treated with TecAn and PenAcAg was altered. The cells lost their characteristic morphology, were shrunk and rounded, were detached from the plate, the cell contact was lost, and they formed islets of more rounded cells in contrast to the untreated cells (Banti et al., 2016, 2021; Kapetana and Banti, 2022; Stathopoulou et al., 2021). Therefore, an apoptotic type of cell death is assumed after the treatment of MCF-7 cells with TecAn and PenAcAg.

Figure 6 Morphological alterations observed in the untreated MCF-7 cells (a), treated with TecAn (b), and PenAcAg (c).
Figure 6

Morphological alterations observed in the untreated MCF-7 cells (a), treated with TecAn (b), and PenAcAg (c).

2.10.5.2 AO/EB staining assay

The apoptotic or necrotic cell death was evaluated with the AO/EB staining assay. The nuclear changes and apoptotic body formation are characteristic of the cascade of apoptosis (Naqvi et al., 2017). AO is a cell permeable fluorescent dye and stains nuclear DNA in both live and dead cells, while EB is a fluorescent dye that only stains nuclear DNA in cells which have lost their membrane integrity (Afsar et al., 2016). In this way, four different cell types, according to the fluorescence emission and the morphological features of the stained nuclei, are observed. (i) viable cells are uniformly stained green, (ii) early apoptotic cells are stained greenish yellow or displayed green yellow fragments, (iii) late apoptotic cells are stained orange or displayed orange fragments, and (iv) necrotic cells show orange to red fluorescing nuclei with no indication of chromatin fragmentation, uniformly red fluorescing, and the cells were swollen to large size (Afsar et al., 2016).

The untreated MCF-7 cells identified by bright uniform green nuclei with organized structures are shown in Figure 7. The percentage of apoptosis and necrosis in the control group was calculated to be 26.8 ± 1.5% and 0%, respectively. In the case of the treated cells with TecAn and PenAcAg, a shrinkage, chromatin condensation, and blebbing of the plasma membrane is observed, indicating that the majority of the cells undergoes apoptotic cell death (Figure 7) (Jaksic, 2012). The percentage of apoptotic and necrotic cells upon treatment with TecAn and PenAcAg are 56.2 ± 0.2% and 0% (TecAn) and 49.3 ± 0.6% and 0% (PenAcAg), respectively. When MCF-7 cells are incubated with cisplatin, the percentage of apoptotic cells rises up to 96.5%. Therefore, TecAn and PenAcAg induce apoptosis but not in the magnitude that cisplatin does.

Figure 7 Fluorescence images of the untreated MCF-7 cells (a), treated with TecAn (b), and PenAcAg (c) for 48 h at 37°C at IC50 values and stained with AO/EB. “L” indicates live cells; “EA” indicates early apoptotic cells; “LA” indicates late apoptotic cells.
Figure 7

Fluorescence images of the untreated MCF-7 cells (a), treated with TecAn (b), and PenAcAg (c) for 48 h at 37°C at IC50 values and stained with AO/EB. “L” indicates live cells; “EA” indicates early apoptotic cells; “LA” indicates late apoptotic cells.

2.11 Ex vivo studies

2.11.1 DNA binding studies

2.11.1.1 UV-Vis absorption spectroscopic studies

In order to examine the binding efficiency of TecAn and PenAcAg towards CT-DNA, UV-Vis absorption spectroscopy was employed (Banti et al., 2016, 2021). There are three types of non-covalent interactions which are studied by UV-Vis spectra: (i) electrostatic interaction with the negatively charged nucleic sugar–phosphates, (ii) groove binding interaction with the grooves of DNA double helix, and (iii) intercalative interaction between the stacked base pairs of native DNA (Banti et al., 2016, 2021). Changes in the configuration of the DNA double helix results in hypochromic and hyperchromic effects; hypochromism shows intercalated or electrostatic binding mode, while hyperchromism is the breakage of hydrogen bonds or groove binding of the compound with the DNA. Moreover, shifting in λ max to higher wavelengths (red shift) suggests stabilization of the helical structure of DNA and shifting of λ max to shorter wavelengths (blue shift) indicates destabilization of the helical structure of DNA (Banti et al., 2016, 2021; Psomas, 2008).

A significant increase in the absorption intensity of DNA solution at λ max = 258 nm, upon treatment with the TecAn and PenAg at various r values (r = [complex]/[DNA]) in a constant [DNA] = 10−4 M is observed (hyperchromism: 13.9% (TecAn) and 9.9% (PenAg)) (Figure 8). This indicates either breakage of hydrogen bonds or groove binding between CT-DNA and TecAn or PenAg.

Figure 8 (I) UV-Vis spectra of CT-DNA in buffer solution in the absence and presence of TecAn (a) and PenAcAg (b) at various r values (0, 0.02, 0.05, 0.07, 0.10, and 0.12; r = [complex]/[DNA], [DNA] = 10−4 M) and (II) plot of A/Ao vs [complex] at 258 nm.
Figure 8

(I) UV-Vis spectra of CT-DNA in buffer solution in the absence and presence of TecAn (a) and PenAcAg (b) at various r values (0, 0.02, 0.05, 0.07, 0.10, and 0.12; r = [complex]/[DNA], [DNA] = 10−4 M) and (II) plot of A/Ao vs [complex] at 258 nm.

The binding constant (K b) of TecAn and PenAg towards CT-DNA was evaluated by monitoring the changes in absorbance of the UV spectra of the agent, ([agent] = 10 μM), at 370–380 nm, with increasing concentration of CT-DNA (Figure 9). K b is obtained from the ratio of the slope to the y intercept in plots [DNA]/(ε A-ε f) vs [DNA], according to the following equation (Banti et al., 2015, 2016; Chrysouli et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021):

(1) [DNA] ( ε A − ε f ) = [DNA] ( ε b − ε f ) + 1 K b ( ε b − ε f )

Figure 9 (I) UV spectra of TecAn (a) and PenAcAg (b) in the absence and presence of CT DNA at r values 1, 0.5, 0.25, 0.17, 0.125, and 0.1 for TecAn and 8.8, 4.4, 2.9, 2.2, and 1.8 for PenAcAg (r = [complex]/[DNA], [complex] = 25 μM (TecAn) and 218 μM (PenAcAg), [CT DNA] = 10–100 μM). (II) Graphical plot of [DNA]/(ε α−ε f) vs [DNA].
Figure 9

(I) UV spectra of TecAn (a) and PenAcAg (b) in the absence and presence of CT DNA at r values 1, 0.5, 0.25, 0.17, 0.125, and 0.1 for TecAn and 8.8, 4.4, 2.9, 2.2, and 1.8 for PenAcAg (r = [complex]/[DNA], [complex] = 25 μM (TecAn) and 218 μM (PenAcAg), [CT DNA] = 10–100 μM). (II) Graphical plot of [DNA]/(ε α−ε f) vs [DNA].

where [DNA] is the concentration of CT-DNA, ε A = A obsd/[compound], ε f is the extinction coefficient for the free compound, and ε b is the extinction coefficient for the compound in the fully bound form (Banti et al., 2015, 2016; Chrysouli et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021). The DNA binding constant (K b) for TecAn is (11.1 ± 0.5) × 104 M−1 and for PenAcAg is (15.6 ± 5.1) × 104 M−1. Moreover, the K b value of tetracycline (TecH2) is (7.2 ± 0.8) × 104 M−1. Therefore, the conjugation of TecH2 and PenH to antimony(v) and silver(i), respectively, form new agents that significantly bind DNA.

2.11.1.2 Fluorescence spectroscopic studies

The binding properties of TecAn and PenAcAg towards DNA were studied further by fluorescent spectroscopy. EB is a strong intercalator of DNA and for this reason it emits intense fluorescent light in the presence of DNA. The quenching of the emitted light when EB is displaced from the CT-DNA-EB complex by an agent indicates an intercalative or minor groove type of binding between DNA and the compound. The CT-DNA-EB complex emits at λ max = 588 nm upon its excitation at λex = 527 nm (Banti et al., 2016, 2021; Kapetana and Banti, 2022; Latsis et al., 2018; Stathopoulou et al., 2021). The fluorescence spectra of the CT-DNA-EB compound with the increasing concentrations of TecAn or PenAcAg (0–600 μM) are shown in Figure 10. The fluorescence emitted from the CT-DNA-EB complex at 588 nm undergoes a 20.3% (TecAn) and 37.4% (PenAcAg) quenching upon increasing the concentrations with respect to the initial fluorescence intensity of the solutions without the agent. The binding constant (K app) was calculated using the equation (Banti et al., 2016, 2021; Kapetana and Banti, 2022; Latsis et al., 2018; Stathopoulou et al., 2021):

(2) K EB [ EB ] = K app [ drug ]

where [drug] is the concentration of the agent at a 50% reduction of the fluorescence, K EB = 107 M−1, and the concentration of [EB] is 2.3 μM. The concentration of the drug at a 50% reduction of the fluorescence is derived from the diagram of I 0/I x vs the concentration of TecAn or PenAcAg (Figure 10), where I 0 and I x are the fluorescence intensities of the CT-DNA in the absence and presence of the compound. K SV is the Stern–Volmer quenching constant and [Q] is the concentration of the quencher (Banti et al., 2016, 2021; Kapetana and Banti, 2022; Latsis et al., 2018; Stathopoulou et al., 2021). The linear Stern–Volmer equation is the following:

(3) I 0 / I = 1 + K SV × [ Q ]

Figure 10 (I) Emission spectrum of CT-DNA-EB complex in the presence of TecAn (a) and PenAcAg (b) ([EB] = 2.3 μM [DNA] = 26 μM [complex] = 0–600 μM) λex = 527 nm. The arrow shows that the intensity changes upon increasing the complex concentration. (II) Inset shows the plots of emission intensity I o/I x vs [complex].
Figure 10

(I) Emission spectrum of CT-DNA-EB complex in the presence of TecAn (a) and PenAcAg (b) ([EB] = 2.3 μM [DNA] = 26 μM [complex] = 0–600 μM) λex = 527 nm. The arrow shows that the intensity changes upon increasing the complex concentration. (II) Inset shows the plots of emission intensity I o/I x vs [complex].

The apparent binding constant K app calculated for TecAn is (1.1 ± 0.2) × 104 M−1, while the corresponding value for PenAcAg is (6.7 ± 1.1) × 104 M−1. In addition, the K app constant of tetracycline (TecH2) is (0.8 ± 0.4) × 104 M−1, TPSb is (5.3 ± 0.5) × 104 M−1, and PenNa is (0.9 ± 0.1) × 104 M−1. These values are lower than the binding constants of the classical intercalators EB (106 M−1), confirming the groove binding of both metallodrugs.

2.11.1.3 Viscosity measurement

The interaction of DNA with an anticancer agent affects the length of DNA which consequently changes the viscosity of its solution (Banti et al., 2021; Kapetana and Banti, 2022; Latsis et al., 2018). Thus, (i) if the agent intercalates with the DNA strands, lengthening of the DNA occurs with an increase in the solution viscosity, (ii) if the agent interacts electrostatically with the DNA, this results in no effect on the DNA length and no significant change in viscosity; (iii) in the case of cleavage of the DNA strands, the length of the DNA decreases along with the viscosity, and (iv) if the agent binds the DNA helix binding covalently with it, decrease in the viscosity is exhibited (Banti et al., 2021; Kapetana and Banti, 2022; Latsis et al., 2018). The relative DNA length (L/L 0) is calculated from the equation:

(4) L / L 0 = ( n / n o ) 1 / 3

where (n/n 0)1/3 shows the relative specific viscosity of the DNA solutions. Solution of CT-DNA (10 mM) is incubated with the increasing amounts of TecAn and PenAcAg. Figure 11 shows the relative specific viscosity (n/n 0)1/3 vs binding ratio. The negligible decrease in the viscosity of DNA solution, upon increasing the concentrations in the case of TecAn, and the less pronounced increase in the case of PenAcAg, suggests a groove binding mode of both metallodrugs (Banti et al., 2021; Kapetana and Banti, 2022; Latsis et al., 2018). This is in agreement with the findings extracted from UV-Vis and fluorescence studies.

Figure 11 Effect of increasing concentrations of TecAn and PenAcAg on the relative viscosity of CT-DNA at 25°C. ([DNA] = 10 mM, r = [compound]/[DNA], n is the viscosity of DNA in the presence of TecAn and PenAcAg and n o is the viscosity of DNA alone).
Figure 11

Effect of increasing concentrations of TecAn and PenAcAg on the relative viscosity of CT-DNA at 25°C. ([DNA] = 10 mM, r = [compound]/[DNA], n is the viscosity of DNA in the presence of TecAn and PenAcAg and n o is the viscosity of DNA alone).

2.12 Study of the peroxidation of linoleic acid by the enzyme LOX

LOX is an enzyme that is mainly distributed in the mitochondrion, and it causes apoptosis. LOX oxidizes linoleic acid to hyperoxo-linoleic acid during the inflammation mechanism. In order to clarify whether the apoptosis caused to the MCF-7 (HD) cells is due to the direct interaction between the agents with DNA or to the mitochondrion dysfunction, which consequently activates the intrinsic pathway of apoptosis through caspases, the LOX inhibitory activity of TecAn or PenAcAg is studied (Banti et al., 2016, 2021; Chrysouli et al., 2018). The hyperoxo-linoleic acid formed is monitored by recording the increase in the absorbance at 234 nm. The degree of LOX activity (A, %) by the influence of TecAn or PenAcAg was calculated by equation:

(5) A ( % ) = 100 × ( u o in the presence of inhibitor ) / ( u o in the absence of inhibitor )

The value of the initial velocity (u 0, mM·s−1) was computed by the formula:

(6) υ o = Δ C /Δ t = Δ A /(Δ t × ε ) = tga/(Δ t × ε )

where C is the concentration of the oxidation product of linoleic acid (hydroperoxy–linoleic acid), t is the reaction time, ε is the molar absorbance coefficient of hydroperoxy–linoleic acid, and tga is the slope of the kinetic curve displayed as absorbance vs time.

Figure 12 shows LOX activity (A%) vs various concentrations of TecAn and PenAcAg. The IC50 value for PenAcAg is 19.8 μM whereas no IC50 value was determined for TecAn at the concentrations tested (up to 60 μM) (Figure 12). For comparison, the corresponding IC50 values of cisplatin is 65.9 μM, while the corresponding values for PenNa and TecH2 are higher than 40 μM (Banti et al., 2016, 2021; Chrysouli et al., 2018; Poyraz et al., 2011). Therefore, a different mechanism of action between TecAn or PenAcAg may be expected.

Figure 12 LOX activity (A%) vs various concentrations of TecAn and PenAcAg.
Figure 12

LOX activity (A%) vs various concentrations of TecAn and PenAcAg.

The reversible or irreversible type of inhibition was investigated by incubating the substrate with the inhibitor before adding the enzyme at various times (Banti et al., 2012). The effect on the enzyme activity of different incubation duration of the substrate with a constant complex concentration supports an irreversible kind of inhibition (Figure S14).

The kind of inhibitor type was evaluated by the steady-state kinetics at different substrate concentrations (ranging from 0.01 to 0.1 mM) in the absence and presence of PenAcAg (19.8 mM). A graphical method with Lineweaver–Burk coordinates (double reciprocal method) was used (Figure 13). From the slope and intercept of the linear graph, the kinetic parameters (K m and V max) were determined. The K m and V max values of free enzyme are 0.035 mM and 27.5 mM·s−1, respectively. The apparent values in the presence of PenAcAg are K m = 0.14 mM and V max = 20.7 mM·s−1 suggesting that PenAcAg inhibits the enzyme via a mixed inhibition mechanism (higher K m value and lower V max) (Xanthopoulou et al., 2008). In this mechanism, both the EI (enzyme-inhibitor) and ESI (enzyme-substrate-inhibitor) complexes are formed (Xanthopoulou et al., 2008). This occurs when the inhibitor binds to a place other than the substrate binding site, resulting in a decrease in catalytic rate and perhaps cell death. These inhibitors could not be used as anti-inflammatory drugs (Xanthopoulou et al., 2008).

Figure 13 Graphical plotting for determination of K m and V max using Lineweaver–Burk coordinates for PenAcAg.
Figure 13

Graphical plotting for determination of K m and V max using Lineweaver–Burk coordinates for PenAcAg.

2.13 Reaction of TecAn with GSH

Antileishmanial drugs include pentavalent antimonials (Sharma et al, 2008). In this case antimony(v) is a pro-drug which is reduced to active antimony(iii) by the tripeptide GSH (Ozturk et al., 2007; Sharma et al, 2008). Sb(v) is biologically less active, while Sb(iii) is the active form of antimonials (Ozturk et al., 2007, 2009, 2010). GSH on the other hand is over-expressed in tumor cells (e.g., its concentration is twice in breast cancer cells than that found in normal ones). It plays a vital role in protecting cancer cells and it is the reason for the resistance they develop against chemotherapy (Banti et al. 2014; Syng-Ai et al., 2004). Therefore, the use of low toxic Sb(v) species which are converted to the active Sb(iii) ones in cancer cells by the GSH is expected to lead to new chemotherapeutics with high selectivity against cancer cells than normal ones. Therefore, the redox reaction between the GSH and TecAn is studied, here, using vibrational spectroscopy The vibrational band of the S–H bond in the ATR-FTIR spectrum of GSH (Figure S15) is absent when it is oxidized by H2O2 (1:1 molar ratio) due to the formation of the disulfide GS-SG. (Figure S15) (Shayani-Jama and Nematollahi, 2010). When H2O2 is replaced by TecAn under the same reaction conditions, the formation of the disulfide with the elimination of the ν(S–H) vibrational band occurs as well (Figure S5) (Kapetana and Banti, 2022).

3 Conclusion

Antibiotics induce cancer cell apoptosis through breakage of DNA strands since DNA is one of their major molecular targets. Thus, the conjugates of tetracycline with antimony(v) TecAn and the corresponding one of penicillin with silver PenAcAg were prepared and tested in vitro against human breast adenocarcinoma cell lines: MCF-7 (positive to hormones receptor (HR+)), MDA-MB-231 (negative to hormones receptor (HR−)). TecAn and PenAcAg inhibit both cell lines in a similar manner implying that hormone receptors play no role in their mechanism of action. Although, TecAn and PenAcAg are classified as toxic agents, according to FDA criteria, they are both significantly more effective against cancerous cells than normal cells compared to cisplatin which, however, is an anticancer drug in clinical use. TecAn and PenAcAg induce apoptosis at 56.2 ± 0.2% (TecAn) and 49.3 ± 0.6% (PenAcAg) of MCF-7 (HD) cells, respectively, like cisplatin, where the percentage of apoptotic cells rises up to 96.5%. Electronic absorption and fluorescent spectroscopic data suggest that both conjugates interact strongly with DNA through groove binding mode. The results are confirmed by the meaningless perturbation in the viscosity of DNA solution when it is treated with TecAn and PenAcAg. GSH readily reduces antimony(v) to highly toxic antimony(iii). TecAn reacts with GSH in a redox reaction where the biological inactive Sb(v) turns in the active Sb(iii). GSH is a cysteine containing tripeptide, over-expressed in the cancer cells. It removes metal-chemotherapeutics from the cytoplasm, preventing their efficient interaction with crucial intracellular components (DNA, mitochondrion, etc.) and developing resistance in cancer cells toward chemotherapy. However, this is cancelled by the active Sb(iii) species formed from the reaction of TecAn with GSH in the cancer cells cytoplasm converting their defending tool to a disadvantage. PenAcAg, on the other hand, inhibits LOX activity stronger than cisplatin suggesting the activation of the apoptosis pathway through mitochondrion.

Experimental

Materials and instruments

All solvents used were of reagent grade and were used with no further purification. Triphenyl antimony, silver nitrate, tetracycline, sodium penicillin, LOX, and linoleic acid were purchased from Aldrich-Merck and they were used without any further purification. DMSO was purchased from Riedel-de Haën. Dulbecco’s modified Eagle’s medium, (DMEM), fetal bovine serum, glutamine, and trypsin were purchased from Gibco, Glasgow, UK. Phosphate buffer saline (PBS), CT-DNA, EB, and propidium iodide were purchased from Sigma-Aldrich. Melting point was measured in open tubes with a STUART SMP30 scientific apparatus, and it is uncorrected. Mid-infrared spectra (4,000–400 cm−1) were obtained on Cary 670 FTIR spectrometer (Agilent Technologies). The fluorescence spectra were recorded on a Jasco FP-8200 Fluorescence Spectrometer. TG–DTA was carried out on a DTG/TG NETZSCH STA 449 C apparatus, under air flow (with a heating rate of 10°C·min−1 (25–500°C). 1H and 13C NMR spectra were recorded with a Bruker AC 400 MHz FT-NMR instrument in DMSO-d 6 solution. A UV-1600 PC series spectrophotometer of VWR was used to obtain electronic absorption spectra. XRF measurement was carried out with Rigaku NEX QC EDXRF analyzer (Austin, TX, USA).

Synthesis of TecAn

0.176 g Ph3Sb(iii) (0.5 mmol) were oxidized by 0.05 mL of H2O2 30% hydrogen peroxide in the presence of 0.222 g tetracycline (0.5 mmol) in 20 mL of Et2O. The solution was stirred in ice bath at 4°C for 5 h. The pale-yellow precipitation was filtered off and dried at ambient conditions. The powder product was dissolved in 20 mL of MeCN followed by filtration to remove the impurities. The clear solution was concentrated to dryness in the rotary evaporator and the solid powder TecAn was washed with 2 mL of Et2O.

TecAn: pale-yellow powder; yield 51%; melting point: 149–154°C; MW = 795.435 g·mol−1. Elemental analysis found: C: 60.10; H: 4.85; N: 3.72; Sb: 14.86 (±0.09)% w/w. Calculated for C40H38N2O8Sb, C = 60.32; H = 4.80; N = 3.52, Sb = 15.29% w/w; ATR-IR (cm−1): 3,052 w, 1,618 m, 1,480 w, 1,432 m, 1,379 w, 1,215 w, 1,175 m, 1,092 m, 1,066 m, 995 m, 857 m, 738 s, 693 s, 618 w, 570 w, 511 w, 451 s, and 414 s. 1H NMR (ppm) in DMSO‑d 6: 9.21 OC(1)NH2, 7.54 C(8)-H, 7.13 C(9)-H, 6.92 C(7)-H, 4.87 C(10)-OH, 2.85 C(10a)-H, 2.5 C(15)-C(16)-H, 1.96 C(11)-H, and 1.51 C(14)-H; 13C NMR (ppm) in DMSO‑d 6: 190.2 (C5), (C3), (C15), 172.5 (C4), 168.9 (C1), 162.9 (C6), 147.7 (C9a), 143.8 (C8), 117.3 (C9), 116.8 (C7), 114.3 (C5a), 103.7 (C4a), 73.3 (C3a), 68.5 (C10), 43.1 (C15), (C16), 34.6 (C10a), 26.4 (C11a), and 23.5 (C11); UV-Vis (DMSO): λ max (logε) = 378 nm (4.21), 304 nm (4.14), and 266 nm (4.11).

Synthesis of PenAcAg

A ddw solution of silver nitrate (0.5 mmol, 0.085 g) was added to a methanolic solution of benzyl-penicillin sodium (PenNa) (0.5 mmol, 0.178 g) and the resulting suspension was stirred for 5 min. The yellowish precipitation was filtered off. The powder product was dissolved in 20 mL of MeCN and the insoluble impurities were removed with centrifugation at 2,000 rpm. The clear supernatant was concentrated to give the PenAcAg.

PenAcAg: yellow powder; yield 20%; melting point: 121–125°C, MW = 964 g·mol−1. Elemental analysis found: C = 44.55; H = 4.43; N = 8.95; S = 6.74; Ag = 19.81 (±0.27)% w/w. Calculated for C36H40Ag2O8N6S2, C = 44.83; H = 4.18; N = 8.71; S = 6.67; Ag = 22.37% w/w; ATR-IR (cm−1): 3,267 m, 2,960 s, 2,927 s, 2,859 s, 1,767 s, 1,659 w, 1,576 m, 1,454 m, 1,394 m, 1,311 m, 1,249 m, 1,088 s, 1,029 m, 890 s, 756 s–m, 697 s, 566 s, and 458 s. 1H NMR (ppm) in DMSO‑d 6: 8.81 [NH–C(6)═O]–H, 7.32–7.22 (Benzyl)-H, 5.48 C(9)-H, 5.40 C(8)-H, 4.14 C(12)-H, 3.55 C(5)-H, 2.09 (MeCN)-H, 1.60 C(16)-H, and 1.48 C(17)-H. UV-Vis (DMSO): λ max (log ε) = 269 nm (3.78).

Biological tests

The biological tests were performed as described previously (Banti et al., 2015, 2016, 2021; Chrysouli et al., 2018; Kapetana and Banti, 2022; Latsis et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021; Tsiatouras et al., 2016).

SRB assay

This study was performed according to the procedure reported previously (Banti et al., 2015, 2016, 2021; Chrysouli et al., 2018; Kapetana and Banti, 2022; Latsis et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021; Tsiatouras et al., 2016).

Evaluation of in vitro genotoxicity with MN assay

The evaluation of genotoxicity caused by TecAn and PenAcAg was performed following the protocol reported elsewhere (Banti et al., 2015, 2016; Kapetana and Banti, 2022; Polychronis et al., 2019; Stathopoulou et al., 2021).

Evaluation of in vivo toxicity with brine shrimp (Artemia salina) assay

Brine shrimp assay was performed as previously reported (Banti and Hadjikakou, 2021).

Cell morphology studies

MCF-7 cells’ morphology was observed under an inverse microscope, as previously reported (Banti et al., 2015, 2016; Kapetana and Banti, 2022; Polychronis et al., 2019; Stathopoulou et al., 2021).

AO/EB staining was used to detect apoptosis: Banti et al. (2015), Kapetana and Banti (2022), and Stathopoulou et al. (2021).

DNA binding studies

UV-Vis studies: this study was performed as described previously (Banti et al., 2015, 2016; Chrysouli et al., 2018; Polychronis et al., 2019; Stathopoulou et al., 2021).

Fluorescence studies: this study was performed as described previously (Banti et al., 2016, 2021; Kapetana and Banti, 2022; Latsis et al., 2018; Stathopoulou et al., 2021).

Viscosity measurements: this study was carried out as previously reported (Banti et al., 2021; Kapetana and Banti, 2022; Latsis et al., 2018).

LOX activity inhibition

This study was performed as previously reported (Xanthopoulou et al., 2006).

Acknowledgments

(a) This work was carried out in partial fulfilment of the requirements for the Master thesis of Ms Z-CMF according to the curriculum of the International Graduate Program in “Biological Inorganic Chemistry,” which operates at the University of Ioannina within the collaboration of the Departments of Chemistry of the Universities of Ioannina, Athens, Thessaloniki, Patras, Crete and the Department of Chemistry of the University of Cyprus (http://bic.chem.uoi.gr/BIC-En/index-en.html) under the supervision of Prof. Sotiris K. Hadjikakou. (b) This work was carried out in partial fulfilment of the requirements for the Master thesis of Ms PZT under the supervision of Prof. Sotiris K. Hadjikakou. (c) PZT acknowledges the financial support from the Eugenides Foundation for a scholarship for her postgraduate studies.

  1. Funding information: Authors state no funding involved.

  2. Author contributions: Paraskevi Z. Trialoni: investigation; Zografia-Christina M. Fyrigou: investigation; Christina N. Banti: investigation, methodology, writing – original draft, and writing – review and editing; Sotiris K. Hadjikakou: conceptualization, methodology, supervision, validation, writing – original draft, and writing – review and editing.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Received: 2022-04-30
Revised: 2022-08-18
Accepted: 2022-07-01
Published Online: 2022-09-07

© 2022 Paraskevi Z. Trialoni et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/mgmc-2022-0016
Keywords for this article
biological inorganic chemistry; organoantimony(V); silver(I); antibiotic; cytotoxicity; DNA–metal complexes interaction
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BY 4.0

Articles in the same Issue

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