Targeting Energy Metabolism by a Platinum(IV) Prodrug as an

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Targeting Energy Metabolism by a Platinum(IV) Prodrug as an Alternative Pathway for Cancer Suppression Suxing Jin,† Yan Guo,‡ Dongfan Song,‡ Zhenzhu Zhu,† Zhenqin Zhang,‡ Yuewen Sun,† Tao Yang,† Zijian Guo,‡ and Xiaoyong Wang*,† †

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, P.R. China State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P.R. China

Inorg. Chem. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 04/24/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Cancer is characterized by abnormal cellular energy metabolism, which preferentially switches to aerobic glycolysis rather than oxidative phosphorylation as a means of glucose metabolism. Many key enzymes involved in the abnormal glycolysis are potential targets of anticancer drugs. Platinum(IV) complexes are potential anticancer prodrugs and kinetically more inert than the platinum(II) counterparts, which offer an opportunity to be modified by functional ligands for activation or targeted delivery. A novel platinum(IV) complex, c,c,t-[Pt(NH3)2Cl2(C10H15N2O3S)(C2HO2Cl2)] (DPB), was designed to explore the effects of axial ligands on the reactivity and bioactivity of the complex as well as on tumor energy metabolism. The complex was characterized by electrospray ionization mass spectrometry and multinuclear (1H, 13C, and 195Pt) NMR spectroscopy. The introduction of dichloroacetate (DCA) markedly increases the lipophilicity, reactivity, and cytotoxicity of the complex and blocks the growth of cancer cells having active glycolysis, and the introduction of biotin (C10H16N2O3S) enhances the tumor-targeting potential of the complex. The cytotoxicity of DPB is increased dramatically in a variety of cancer cell lines as compared with the platinum(IV) complex PB without the DCA group. DPB alters the mitochondrial membrane potential and disrupts the mitochondrial morphology. The levels of mitochondrial and cellular reactive oxygen species are also decreased. Furthermore, the mitochondrial function of tumor cells was impaired by DPB, leading to the inhibition of both glycolysis and glucose oxidation and finally to the death of cancer cells via a mitochondria-mediated apoptotic pathway. These findings demonstrate that DPB suppresses cancer cells mainly through altering metabolic pathways and highlight the importance of dual-targeting for the efficacy of anticancer drugs.

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INTRODUCTION Platinum-based anticancer drugs represented by cisplatin play important roles in the clinical chemotherapy of cancers.1,2 These drugs usually bind to purine bases through covalent bonds to form bifunctional intra- or interstrand DNA adducts, which inhibit DNA replication and lead to cell death.3 However, systemic toxicity and drug resistance have hampered the clinical application of these drugs.4,5 To obtain novel platinum anticancer drugs with ideal therapeutic effects, creative strategies keep emerging in recent years. PtIV complexes are attractive prodrugs for overcoming the drawbacks of PtII complexes due to their diversified activities and low systemic toxicity.6 Moreover, they are more kinetically inert and less reactive than their PtII precursors and are convenient for oral administration.7 Once inside cancer cells, PtIV complexes would be reduced by cellular reductants to release two axial ligands and a cytotoxic PtII moiety, forming Pt-DNA adducts and interfering with DNA replication and transcription.8 The axial ligands could confer favorable pharmacological properties to the PtIV complex, including enhanced lipophilicity, increased solubility, modified rates of © XXXX American Chemical Society

reduction, targeting ability, and incorporation of additional bioactive molecules.9,10 The abnormal energy metabolic mode is one of the characteristics of tumor cells, in which glucose is converted to lactic acid even in the presence of oxygen, known as aerobic glycolysis or “Warburg effect”, rather than metabolized through mitochondrial oxidative phosphorylation (OXPHOS) in normal cells.11 The mitochondrion is the center for energy and substance metabolisms in eukaryotic cells and is crucial for cell proliferation, free radical production, and apoptosis.12 The unique glucose metabolic pathway of cancer cells presents a promising therapeutic target for anticancer drugs, and some studies have aimed at glycolysis in cancer treatment.13 Pyruvate dehydrogenase kinases (PDKs) that are overexpressed in cancer cells are key enzymes in the glucose metabolic pathway responsible for blocking the oxidative decarboxylation of pyruvate to meet the high demand of oxygen, while inhibition of PDKs can increase the activity of Received: March 12, 2019

A

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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the apoptosis of cancer cells; moreover, it may help to evade the off-target effects frequently encountered in chemotherapy.

pyruvate dehydrogenase complex (PDC) and balance the consumption and supply of oxygen, leading to cell death.14 The orally available dichloroacetate (DCA) is an inhibitor of glycolysis that activates OXPHOS through inhibiting PDK, thereby increasing the amount of pyruvate entering into mitochondria and promoting glucose oxidation rather than glycolysis.15 It has been shown that DCA reverses mitochondrial changes in various cancers and promotes tumor cell apoptosis in a mitochondrial-mediated apoptotic pathway.16 A PtIV prodrug mitaplatin containing DCA moieties in the axial positions has displayed a dual-killing mode toward cancer cells,17 that is, damaging nuclear DNA by the platinum center and attacking mitochondria by the released DCA upon reduction; however, it has no active tumor-targeting ability. The strategy of targeting mitochondrial metabolism has been widely attempted, but some challenges, such as simultaneous targeting at tumor tissues and mitochondria, still exist. Since mitochondria are omnipresent in various cells, how to avoid damage to mitochondria of normal cells becomes a complicated problem. Biotin (vitamin H or B7) is favorably integrated by tumor cells owing to the overexpression of biotin-specific uptake systems on the surface of malignant cells and its high affinity for avidin proteins;18−20 therefore, it is often adopted as a tumor-targeting moiety in drug delivery. Recently we introduced biotin to a PtIV complex, obtaining a monobiotinylated complex (Pt-Bio-I) and a dibiotinylated one (Pt-Bio-II).21 These complexes are highly cytotoxic against breast cancer cells; particularly, Pt-Bio-I is more readily reduced and hence more reactive to DNA than Pt-Bio-II. We herein report the biological properties of a novel PtIV complex c,c,t-[Pt(NH3)2Cl2(biotin)(DCA)] (DPB, Figure 1)

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RESULTS AND DISCUSSION Synthesis and Characterization. PtIV complexes are commonly obtained by oxidizing PtII precursors with hydrogen peroxide or chlorine and subsequent conjugating with axial ligands, resulting in chloride, hydroxyl, or carboxyl groups staying in the axial position(s).23 DPB was synthesized by introducing DCA and biotin moieties to the axial positions of the PtIV center. The reference complex PB was synthesized according to our previous report.21 All of the complexes were fully characterized by 1H-, 13C-, 195Pt-NMR, ESI-MS, ESIHRMS, and elemental analysis (Figures S1−S3 and Table S1). DPB and PB are soluble in a range of organic solvents such as methanol, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), but are almost insoluble in water. DPB carries both a tumor-targeting group biotin and a mitochondrion-targeting group DCA, which are expected to assist the compound to preferentially reach tumor cells and mitochondria consecutively in vivo. Lipophilicity and Stability. Lipophilicity is an important parameter for predicting the membrane penetration ability of a compound.24 We thus tested the lipophilicity of DPB and PB by measuring their partition between water and n-octanol using the shake-flask method. The log PO/W of DPB and PB is −0.55 ± 0.04 and −1.47 ± 0.08, respectively, which is larger than that of cisplatin (−2.3),25 showing that the lipophilicity of these complexes follows an order of DPB > PB > cisplatin. The results suggest that DPB may penetrate the lipid bilayer of the cellular membrane and accumulate in tumor cells more readily than cisplatin (vide infra). DPB is stable over 48 h even in a highly acidic environment (pH 4.0, Figure S4), suggesting that it might be administered orally in clinical treatments. Reduction Kinetics. PtIV complexes can be reduced to PtII analogues during intracellular reaction with glutathione (GSH) or ascorbic acid (AsA),26 which would influence the biological activities of the complexes.27 Therefore, we monitored the reductions of PB and DPB in the presence of AsA at 37 °C in the dark for several days by 195Pt NMR spectrometry. The peak for PtIV vanishes and that for PtII appears in the spectra as the time extends, indicating the complexes were reduced; however, the reduction rate of PB is quite faster than that of DPB, as PB was reduced almost completely, while DPB just started to be reduced after 6 h, and DPB did not disappear until 36 h later (Figure S5). The reduction of PtIV complexes is dependent on the nature of equatorial and axial ligand(s).28 PtIV complexes with a monohydroxido or monocarboxylato framework may have advantages over those with either a

Figure 1. Chemical structures of PB and DPB.

which exhibits multifacet antitumor properties. DPB easily accumulates in cancer cells and influences the mitochondrial energy metabolism of tumor cells after reduction, affecting the expression of several key proteins and interacting with DNA. The introduction of different functional moieties, especially DCA, enriches the reactivity and bioactivity of the complex. Compared with DCA and PB (formerly Pt-Bio-I) containing only a tumor-targeting group,21,22 DPB affects the energy metabolism of cancer cells at low concentrations and promotes

Table 1. IC50 Values (μM) of PB and DPB toward HeLa, HepG2, HCT-116 Cancer Cells, and L-02 Normal Cells at 72 h Determined by the NRU Assay, with Those of Cisplatin, Oxoplatin, DCA, and Biotin as References cell line biotin (±) PB DPB cisplatin oxoplatin DCA biotin

HeLa

HepG2

+++ 5.16 1.97 1.44 8.25 >128.00 >128.00

± ± ± ±

HCT-116 −

+++ 0.79 0.29 0.40 0.36

6.62 1.86 1.42 5.74 >128.00 >128.00

L-02

± ± ± ±

0.30 0.74 0.19 0.82

B

14.62 18.60 3.07 16.93 >128.00 >128.00

± ± ± ±

0.52 1.13 0.38 1.36

3.30 13.24 3.35 25.52 >128.00 >128.00

± ± ± ±

0.63 0.44 0.55 1.50

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry dihydroxido or dicarboxylato framework.28b The result is consistent with our previous findings.29 Cytotoxicity and Apoptosis. The antiproliferative properties of PB, DPB, and cisplatin were evaluated in the human cervical cancer (HeLa) and liver cancer (HepG2) cell lines expressing biotin receptor (biotin+) and the human colorectal cancer (HCT-116) cell line without biotin receptor (biotin−) as well as the normal human liver (L-02) cell line using the neutral red uptake (NRU) assay. Cisplatin, oxoplatin, DCA, and biotin were used as references. The half maximal inhibitory concentrations (IC50) of the complexes at 72 h are summarized in Table 1. The IC50 values of DPB toward HeLa and HepG2 cells at 72 h are comparable to those of cisplatin. The biotin+ cancer cells are more sensitive to PB and DPB than the biotin− cells, suggesting that PB and DPB may preferentially accumulate in these cancer cells because of the biotin ligand.18 However, similar to oxoplatin, PB is less cytotoxic than DPB against the biotin+ cancer cell lines, although its reduction rate is faster than that of DPB (vide supra). This incongruity may be attributed to its lack of DCA ligand. In addition, the less lipophilicity of PB may also account for its lower cytotoxicity. The IC50 value of DPB for L-02 cell line is much higher than that of cisplatin, implying that DPB is less toxic to normal cells. Although DCA is almost nontoxic under the same conditions, it greatly enhanced the cytotoxicity of DPB toward cancer cells. It seems that the lipophilicity and ligands of the complex, along with the expression of biotin receptor and attribute of the cells (cancerous vs normal), influence the cytotoxicity of these complexes. The pathway of cell death induced by the complexes was investigated using flow cytometry. HeLa cells were treated with each complex for 72 h and stained with Annexin V-FITC and PI to evaluate the percentage of live (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI +), and necrotic (Annexin V−/PI+) cells. The results showed that all the complexes can induce apoptosis as compared to the untreated control cells. DPB mainly induced the cells to undergo late apoptosis at its IC50 (2 μM), which is similar to cisplatin, but the effect was much weaker (Figure S6). These apoptotic results are basically consistent with the trend of cytotoxicity, which highlight the prodrug nature of PtIV complexes, that is, they need some time to be reduced to active PtII species. Cellular Uptake. In order to investigate the relationship between the cellular uptake and the cytotoxicity of the complexes, the intracellular accumulation and distribution of the complexes in terms of Pt were evaluated by ICP-MS. The Pt content in different cellular components of HeLa cells is shown in Figure 2. The total Pt content is inconsistent with the cytotoxicity; for example, DPB has the largest total Pt content, but its cytotoxicity is weaker than cisplatin due to its prodrug trait. We further determined the Pt content in different fractions of the cells. The results showed that a considerable amount of Pt is trapped in the cell membrane for DPB. The actual accumulation of Pt in cells should be the remaining Pt after subtraction of the “viscous” Pt. Therefore, the Pt content inside HeLa cells is 3.38 ± 0.08, 3.50 ± 0.07, and 6.55 ± 0.12 ng per μg whole protein for cisplatin, PB, and DPB, respectively, which corroborate the favorable effect of lipophilicity on the cellular uptake of PB and DPB. Interestingly, although the total Pt content for DPB is significantly higher than that of PB, nearly half was stuck in the cell membrane. It seems that the complex with larger bulk is easily stuck in the

Figure 2. Pt accumulation in HeLa cells after treatment with 1 μM of PB, DPB, and cisplatin, respectively, for 72 h.

cell membrane. Of note, the Pt content in the nucleus for DPB is much lower than that for cisplatin (0.58 ± 0.17 vs 1.22 ± 0.12 ng/μg whole protein), which may weaken its damage to nuclear DNA as compared to cisplatin. To further investigate whether the complexes can target mitochondria, the Pt content in the mitochondria of HeLa cells (ng/μg protein) after incubation with PB, DPB, and cisplatin (1 μM), respectively, for 72 h was determined by ICP-MS using a mitochondria isolation kit. Among the complexes, DPB showed the highest Pt accumulation in mitochondria (2.33 ± 0.04), followed by cisplatin (1.53 ± 0.05) and PB (1.07 ± 0.02), suggesting that DPB can target mitochondria in cancer cells, which could cause damage to mitochondrial DNA and mitochondrial functions. DNA Platination. The interactions of DPB with calf thymus DNA (CT-DNA) in the absence and presence of AsA were first investigated by CD spectrometry to check its reactivity to DNA. It is known that PtII complexes can bind to DNA and change its conformation, while PtIV complexes cannot. Our test confirmed that DPB cannot directly interact with CT-DNA; in the presence of AsA, however, the maximum ellipticity of both the positive and negative bands of DNA increased (Figure S7), thus manifesting that DPB can unwind the DNA helix and change the B-DNA to A-DNA to some extent after reduction to PtII species. The platination of cellular DNA was monitored after HeLa cells were exposed to PB, DPB, and cisplatin for 48 and 72 h, respectively. As shown in Figure 3, the level of DNA platination follows an order of PB < DPB < cisplatin, which is consistent with their nuclear accumulation and corresponding cytotoxicity (Table 1). In other words, the platination of cellular DNA is positively related to the cytotoxicity of the complex. Impressively, the level of DNA platination induced by PB is markedly lower than that induced by DPB, though they accumulate similarly in the nucleus. The result suggests that some non-nuclear DNA, particularly mitochondrial DNA, participated in the formation of DNA adducts. Cell Cycle. The cell cycle of HeLa cells after treatment with cisplatin, PB, and DPB, respectively, was examined by flow cytometry after staining DNA with PI at 48 h. As shown in Figure 4, these complexes induced totally different cell cycle distributions. DPB arrested the cells mainly in the G2 phase, raising the cell distribution from 9% to 67% in comparison with the control group, while cisplatin mainly arrested the cells in the S phase. DCA almost has no effect on the cell cycle at C

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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relative sensitivity of PDKs to DCA inhibition is PDK2 ≈ PDK4 > PDK1 ≫ PDK3.33 We hence examined the expression of PDK2 by Western blotting. As shown in Figure 5, the

Figure 5. Expression of PDK2 in HeLa cells after treatment with cisplatin, PB, and DPB for 48 h.

expression of PDK2 in the DPB-treated HeLa cells was markedly inhibited as compared with that in the PB- and cisplatin-treated cells, and the inhibition is positively related to the concentration of the complex. The result suggests that DPB can increase the activity of PDH by inhibiting PDK, interfere with the energy metabolism of HeLa cells, and decrease MMP hyperpolarization, which would open the mitochondrial transition pores (MTPs).32 Mitochondrial Morphology and Mitochondrial Membrane Potential (MMP). The occurrence of tumors not only relates to the abnormal proliferation and differentiation of cells but also to the abnormal apoptosis. Apoptosis mainly includes an exogenous or extrinsic pathway mediated by death receptors and an endogenous or intrinsic pathway mediated by mitochondria.34 Mitochondria produce most of the cell energy, generate reactive oxygen species (ROS) as byproducts, and regulate apoptosis through a mitochondrial permeation transition pore (mtPTP).35 As dynamic networks, mitochondria often change their shapes and subcellular distribution.36 Since mitochondrial morphology reflects the balance between continuous and antagonistic fusion and fission reactions,37 we examined the mitochondrial ultrastructure of HeLa cells using transmission electron microscopy (TEM). As shown in Figure 6, the mitochondria in untreated cells are predominantly round and lack the crystal pattern of normal mitochondria. These abnormalities are similar to those observed in Morris hepatoma mitochondria in some ways,38 suggesting that the OXPHOS of HeLa cells is abnormal, and the malignancy of the cells is positively related to the abnormality of mitochondrial morphology.37,39 As compared with the control cells, the morphology of mitochondria in DPB-treated cells changed significantly, and a large number of vacuoles appeared in the cells, quite like the phenomenon of early autophagy.39 Some mitochondrial membranes disappeared, and some cristae were damaged with distortion. By contrast, the mitochondria of PBor cisplatin-treated cells only show subtle or mild changes. These results imply that the destructive changes in mitochondrial morphology may affect MMP and energy metabolism of the cells. In order to determine whether DPB could disrupt the integrity of the mitochondrial membrane, MMP was measured using the fluorescent probe JC-1 (5,5,6,6′-tetrachloro-1,1′,3,3′tetraethyl-imidacarbocyanine iodide). JC-1 accumulates in the matrix of mitochondria to form a polymer (J-aggregates) and gives off red fluorescence when MMP is high, while it exists as a monomer to produce green fluorescence when MMP is low.40 We found that incubation of HeLa cells with DPB induced an evident increase in green fluorescence as compared with the control group (Figure S9), thus suggesting a decrease

Figure 3. Platination of cellular DNA (pg Pt/ng DNA) in HeLa cells induced by PB, DPB, and cisplatin (1 μM), respectively, after incubation for 48 and 72 h.

Figure 4. Impact of PB, DPB, and cisplatin (1 μM) on the cell cycle of HeLa cells after incubation for 48 h.

the same concentration (Figure S8) as compared to normal cells, which means that it is nontoxic to the cells and does not affect the cell cycle at the tested concentration, but it does affect the cytotoxicity or cell cycle arrest once it was introduced to the axial position of PtIV center. The difference in cell cycle arrest between DPB and cisplatin strongly suggests that the antiproliferative mechanism of DPB differs from that of cisplatin. Of course, other factors such as mitochondrial dysfunction may also be involved in the cell cycle process (vide infra). Inhibition of PDK. Increased aerobic glycolysis and oxidative stress are important metabolic features of cancer cells. Hence, interfering with aerobic glycolysis is an alternative strategy for the treatment of cancers, in which pyruvate dehydrogenase (PDH), the key regulator of cellular metabolism, is inhibited by PDK.30 As a structural analog of pyruvate, DCA can down-regulate the PDK activity, thereby reducing the inhibition to PDH; the abnormal metabolism of cancer cells is thus reversed from glycolysis to OXPHOS, resulting in the suppression of tumor growth in vitro and in vivo.31 PDK2 is the most ubiquitous kinase among the four isoforms of PDK and is most susceptible to the inhibition by DCA.32 The D

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 6. TEM images of mitochondria in HeLa cells after treatment with 1 μM of PB, DPB, and cisplatin, respectively, for 48 h.

of MMP. Furthermore, the fluorescence intensity ratio of green to red (G/R) that reflects the ability of the complex to depolarize mitochondria was also determined. The JC-1stained HeLa cells showed a dramatic decrease in MMP for the DPB-treated cells (G/R = 3.38 ± 0.40) as compared with the control (G/R = 0.65 ± 0.21), cisplatin- (G/R = 1.85 ± 0.14), or PB-treated cells (G/R = 1.79 ± 0.46) at the same Pt concentration. These findings suggest that DPB could influence the function of mitochondria by reducing the MMP of HeLa cells. It is known that the decrease of MMP induced by DCA is associated with the production of mitochondrial ROS;33 therefore, the decrease of MMP induced by DPB may result from the production of ROS because of the DCA ligand. Generation of ROS. Mitochondrial membrane depolarization is related to the production of ROS, which play important roles in apoptosis in various cancer cells.41 Since DPB can remarkably reduce MMP, we suppose it could induce mitochondria to produce ROS. Intracellular ROS levels in HeLa cells in the presence of PB, DPB, and cisplatin were thus monitored using the ROS-responsive fluorescent probe 2′,7′dichlorofluoresceindiacetate (DCFH-DA). We found that the generation of ROS induced by DPB increased evidently in the first 36 h (Figure 7A) and is comparable to that induced by cisplatin at 48 h (Figure 7B). In contrast, PB had a weaker impact on the untreated cells (Figures 7B and S10). These results reinforce our assumption that the reduction in MMP and severe deformation of mitochondria in HeLa cells are related to the increased production of ROS. Effect on Mitochondrial Bioenergetics. Normal OXPHOS activity, homeostatic ROS, and MMP are key factors for inducing apoptosis via the mitochondrial pathway.42 However, OXPHOS is largely defective in cancer cells, and the glycolytic pathway is commonly used to produce ATP. This unique metabolic mode makes mitochondrion a major target for cancer treatment. Glycolysis in the absence of OXPHOS in cancer cells causes an increase in lactic acid; therefore, the level of extracellular acid indirectly reflects the glycolytic capability of cells. The extracellular acidification rate (ECAR) in response to sequential addition of D-glucose, oligomycin (an ATP synthase inhibitor), and 2-deoxy-D-glucose (2-DG, a hexoki-

Figure 7. Generation of ROS in HeLa cells after treatment with DPB (1 μM) for 24, 36, and 48 h, respectively (A) and that after treatment with PB, DPB and cisplatin (1 μM), respectively, for 48 h (B) measured by using DCFH-DA in combination with flow cytometry.

nase inhibitor) to HeLa cells was determined to assess the glycolysis, maximal glycolytic, and glycolytic reserve capacities in the presence of DPB. The overall results are shown in Figure 8A. Oligomycin triggered different changes in lactic acid production for the DPB-treated and untreated cells, in that the ECAR for the former is obviously lower than that for the latter. 2-DG induced a rapid decline in the ECAR for both DPBtreated and untreated cells. The specific changes in ECAR of HeLa cells with or without DPB are shown in Figure 8B. The results indicate that DPB can significantly weaken the maximum glycolytic capacity and glycolytic reserve capacity of HeLa cells. Therefore, DPB is an effective inhibitor of glycolysis in HeLa cells. To determine whether DPB affects the mitochondrial oxidative respiration in cancer cells, we measured the oxygen consumption rate (OCR) in HeLa cells, which reflects the status of basal respiration, ATP synthesis, max respiration, and spare respiratory capacity during OXPHOS. The overall profiles of OCR are shown in Figure 8C, and specific changes of OCR in responding to different stimuli are shown in Figure 8D. The basal respiration for DPB-treated cells decreased significantly as compared to that for control cells, indicating E

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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the metabolic efficiency of mitochondria was reduced. Subsequently, addition of oligomycin attenuated the OCR of ATP synthesis more evidently in DPB-treated cells than in control cells. The maximal respiratory capacity was markedly reduced for the DPB-treated cells after the injection of mitochondrial uncoupler FCCP into the media. In addition, injection of a combination of antimycin A (an inhibitor of mitochondrial complex III) and rotenone (an inhibitor of mitochondrial complex I) also significantly inhibited the respiration in DPB-treated cells. Figure 8E presents the absolute OCR and ECAR values in the same energy map, which indicates that DPB can inhibit both the glycolysis and mitochondrial respiration in HeLa cells. Figure 8F shows that HeLa cells have a high OCR/ECAR ratio, suggesting that the fast proliferating cells need more effective metabolism to meet the energy requirement;43 the OCR/ECAR ratio decreased drastically for the DPB-treated cells, implying that DPB could inhibit the cell proliferation through suppressing the energy metabolism. Furthermore, we evaluated the mitochondrial dehydrogenase activity of HeLa cells in the presence of PB and DPB, respectively, by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) assay, which could reflect the mitochondrial metabolic activity by converting the colorless tetrazolium to the purple formazan dye.44 The optical density (OD) of the purple formazan solution is 2.17 ± 0.13 and 1.51 ± 0.25 after the treatment with PB and DPB for 72 h, respectively. The smaller OD for the DPB-treated cell solution suggests that the mitochondrial metabolic activity was impaired more severely by DPB than by PB, which is consistent with their cytotoxicity. Interestingly, it is known that MTT is mainly reduced by the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) and the glycolytic enzyme of endoplasmic reticulum in the cell viability test;45 thus, the reduction of MTT can be regarded as an indicator for the production of glycolytic NADPH. The smaller OD implies

Figure 8. Glycolytic (A and B) and respiratory (C and D) profiles of untreated and DPB-treated HeLa cells at 36 h; the OCR of basal respiration from the Mito stress test and the glycolysis-related ECAR from the glycolysis stress test, respectively (E); and OCR/ECAR ratios in HeLa cells treated with or without DPB (F). Three replicate experiments prepared from a single-cell culture were measured independently.

Figure 9. Expressions of Bax, Bad, Bcl-2, Cyto c, caspase-3, caspase-9, and p53 in HeLa cells after treatment with cisplatin, PB, and DPB for 48 h (A) and the ratios of proteins to α-tubulin (B and C), respectively. F

DOI: 10.1021/acs.inorgchem.9b00708 Inorg. Chem. XXXX, XXX, XXX−XXX

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the PtIV scaffold could remarkably increase the cellular Pt accumulation and enhance the therapeutic superiority over cisplatin in the therapy of breast cancers. In order to exert influence on the abnormal energy metabolism of cancer cells, a multifunctional PtIV prodrug DPB was designed in this study. Apart from the tumor-targeting potential derived from biotin, DBP also possesses the capability to intervene in the glycolysis of cancer cells owing to the introduction of DCA. As a result, the lipophilicity and reactivity of DPB are increased, and the energy metabolic efficiency of the cells is suppressed, leading the cancer cells to die through an alternative mitochondrial pathway besides DNA damage. Particularly, the newly bestowed properties of DPB, including activation of PDH, depolarization of MMP, promotion of ROS, deformation of mitochondria, and inhibition to OXPHOS and glycolysis significantly interfered with the energy metabolism, starving the cancer cells to death. Thus, the action of DPB represents a new mechanism for platinum-based anticancer drugs, which may minimize the detrimental effects on normal cells and improve the curative effect on cancers.

that the production of glycolytic enzymes in HeLa cells was more reduced by DPB than by PB, which further confirms that DPB can inhibit both the mitochondrial and glycolytic metabolic activities of cancer cells. Mitochondrion-Mediated Apoptosis. We further investigated the proteins potentially involved in the DPB-induced apoptosis. Bcl-2 family proteins, including antiapoptotic (Bcl2) and proapoptotic (Bax, Bad) proteins, play important roles in the mitochondrion-induced apoptosis, and the translocation of Bax on the outer membrane of mitochondria can lead to the decrease of MMP and the release of cytochrome c (Cyto c) into the cytoplasm.46 To ascertain whether these proteins are influenced by DPB, the expressions of Bax, Bad, Bcl-2, and Cyto c in HeLa cells were determined by Western blot. As shown in Figure 9A,B, the expressions of Bax, Bad, and Cyto c increased after incubation with DPB for 48 h, while that of Bcl2 decreased significantly. Cyto c released from mitochondria after stimulation by Bax can activate apoptotic protease activating factor-1 (Apaf-1); the activated Apaft-1 further activates caspase-9, which cleaves caspase-3 zymogen, leading to apoptosis.47 Hence, the expressions of caspase-9 and -3 in HeLa cells were determined. As shown in Figure 9C, caspase-9 and -3 are increased by DPB, thus suggesting that DPB can promote apoptosis through this way. The tumor suppressor gene p53 is a negative regulator in the cell growth cycle, which is related to the regulation of cell proliferation and apoptosis as well as mitochondrial respiration. Deletion of p53 can increase the level of glycolysis and reduce aerobic respiration.48 The expression of p53 in HeLa cells in the presence of DPB was hence tested. As shown in Figure 9A,C, the level of p53 was enhanced markedly after the treatment with DPB, thus demonstrating that the DPBinduced apoptosis has a close relation with the mitochondrionassociated proteins. All of the above results indicate that the apoptosis of HeLa cells induced by DPB is related to the mitochondrial pathway as summarized in Figure 10.

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EXPERIMENTAL SECTION

Synthesis of Compounds. Oxoplatin was synthesized by oxidizing cisplatin with 30% H2O2 as reported in the literature.49 Yield: 67.8%. PB (formerly Pt-Bio-I) was synthesized as we described previously.21 DPB was prepared using a published method with some modifications.50 Specifically, dichloroacetic anhydride (1.20 g, 5 mmol) was added to the solution of PB (0.140 g, 0.25 mmol) in anhydrous dichloromethane (DCM, 10 mL) under nitrogen protection. After stirring at room temperature for 12 h, a suspension was obtained. The filtrate was collected after filtration, and the crude product was washed with diethyl ether several times and then dried in vacuum as a white powder. Yield: 90 mg, 53.90%. 1H NMR (DMSOd6, 600 MHz) δ (ppm): 1.31−1.51 (m, 6H, −CH2−), 2.24 (t, 2H, −CH2−COO), 2.57 (d, 1H, CH2−S), 2.81 (dd, 1H, CH2−S), 3.10 (m, 1H, −CH−S), 4.14 (m, 1H, N−CH−C), 4.30 (m, 1H, N−CH− C), 6.37−6.42 (d, 2H, NH−CO), 6.43 (d, 1H, CHCl2−CO), 6.48− 6.68 (m, 6H, NH3). 13C NMR (DMSO-d6, 101 MHz) δ (ppm): 26.13, 28.60, 28.76, 36.75, 55.94, 59.67, 61.50, 163.34, 181.54. 195PtNMR (DMSO-d6, 86 MHz) δ (ppm): 1054.43. ESI-MS (negative mode, m/z) found (calcd) for [Pt(NH3)2Cl2(C10H15N2O3S)(C2HO2Cl2) + H]−: 669.08 (668.97). HR-ESI-MS (negative mode, m/z): see Figure S3. Elemental analysis determined (calcd) for DPB: C, 20.48 (21.47); H, 3.38 (3.30); N, 8.31 (8.35). Lipophilicity and Stability. The lipophilicity (log PO/W) of PB and DPB was measured using the shake-flask method in the 1octanol/buffer system. An equal volume of PB and DPB solution (1.0 mL) was mixed with 1-octanol and placed in the rotary mixer (25 ± 0.1 °C) at 200 rpm for 4 h. Centrifugation was carried out at 2500 rpm for 15 min to separate different phases. The solute in the aqueous phase was separated, and the Pt content in initial and final aqueous phases was determined by ICP-MS. The log PO/W was calculated using the following equation:

Figure 10. Mitochondrial pathway for the DPB-induced apoptosis in HeLa cells.

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log PO/W = log [([C]initial −[C]final )/[C]final ]

CONCLUSION Abnormal energy metabolism is a ubiquitous feature of cancer cells. The energy supply of cancer cells largely relies on glycolysis, whereas normal cells initiate glycolysis only in the absence of oxygen. Targeting this unique metabolic pathway can selectively destroy cancer cells without affecting normal cells. Drugs that target glycolysis-related enzymes or proteins of cancer cells can induce apoptosis through arresting cell cycles, thereby providing a new direction for chemotherapy. In our previous work, we found that tethering biotin moieties to

DPB was dissolved at 1 × 10−5 M in DMEM containing 0.1% DMSO. UV−vis absorption spectra of DPB were recorded on a Specord 200 UV−vis spectrophotometer at 37 °C and pHs 4.0, 7.4, and 8.0, respectively. Reduction Kinetics. The samples were prepared by reacting PB and DPB (2 mM) with 2 equiv of AsA in 70% DMSO/30% D2O solutions, respectively. The time course of the reaction was monitored by 195Pt-NMR after the samples were incubated at 37 °C for 0, 6, 12, 24, 36, and 48 h, respectively, in the dark. Cytotoxicity and Apoptosis. The HeLa, HepG2, HCT-116, and L-02 cells were cultivated overnight in DMEM supplemented with G

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Inorganic Chemistry 10% heat-inactivated fetal bovine serum and 100 U mL−1 penicillin in an incubator under 5% CO2 at 37 °C and plated in 96-well plates with 2 × 103 cells per well. PB and DPB were dissolved in DMSO, respectively, and diluted with culture medium to make the concentration of DMSO lower than 0.5%, while a stock solution of cisplatin was dissolved in PBS. The stock solutions of PB, DPB, and cisplatin were diluted with a complete medium and then added into the wells. After the cells were incubated for 72 h, neutral red solution (20 μL) was added to each well and incubated for 2 h. The medium was removed, the samples were washed with PBS solution, and then the cell lysis solution (200 μL) was added to the cells. The absorbance of the solution was recorded on an ELISA plate reader at 540 and 690 nm in order to get a more exact value by correcting for background signals. The background OD measured at 690 nm was subtracted from the total OD at 540 nm. Each test was performed in triplicate. The cell viability (%) was calculated using the following equation:

were collected by trypsinization and washed with PBS, fixed in icecold ethanol (70%) for 12 h, pelleted by centrifugation, stained with PI in PBS for 30 min, and analyzed by flow cytometry using FACS. Mitochondrial Morphology and MMP. HeLa cells (1 × 106) were treated with 1 μM of PB, DPB, and cisplatin, respectively, for 48 h. The cell samples were prepared, stained with uranyl acetate and lead citrate, collected on copper grids, and subjected to taking TEM images. HeLa cells were cultured at a density of 1 × 106 cells mL−1 and allowed to grow overnight at 37 °C. Cells were treated with cisplatin, PB, and DPB (1 μM) for 48 h at 37 °C. A solution of JC-1 (10 μg mL−1 in DMEM) was added and incubated at 37 °C for 20 min. The cells were isolated and washed three times with JC-1 staining buffer by centrifugation (1800 rpm, 3 min, 4 °C). The fluorescence of green channel was excited at 488 nm, and the emission was collected at 510−545 nm. The fluorescence of red channel was excited at 525 nm, and the emission was collected at 575−630 nm. Quantification of fluorescence intensities and the ratio of green to red fluorescence were carried out using Zen 2.3. Determination of ROS. HeLa cells were seeded in 6-well micro plates at a density of 2 × 105 cells per well and cultivated for 24 h. The cells were treated with cisplatin, PB, and DPB (1 μM) for 36, 48, and 72 h, respectively, and then were washed with PBS (1 mL × 2) and resuspended in serum-free medium. The cells were incubated with DCFH-DA (10 μM) at 37 °C for 30 min or with MitiSOX (5 μM) for 10 min in the dark and washed twice with PBS. The fluorescence change in cells was measured by flow cytometry (λex = 485 nm, λem = 520 nm). Western Immunoblot Analysis. HeLa cells were seeded in 6 cm plates and treated with cisplatin, PB (1 μM), and DPB (1, 5 μM) for 48 h, respectively. The suspended and attached cells were collected and lysed by ice-cold RIPA buffer with protease and phosphatase inhibitor. The cell lysates were incubated on ice for 20 min and centrifuged at 12,000 g for 15 min at 4 °C. Protein concentrations were measured using protein assay reagents, and equal amounts of protein per lane were separated on SDS-PAGE gel and transferred to a PVDF membrane. The membrane was incubated with monoclonal anti-Cyto c, PDK2, Bad, Bax, Bcl-2, caspase-3, caspase-9, p53, and αtubulin, followed by incubation with the peroxidase-labeled goat antirabbit HRP secondary antibody. Western blots were visualized by enhanced chemiluminescence detection system. Relative grayscale was calculated by ImageJ. Mitochondrial Bioenergetics. ECAR and OCR were tested on Seahorse XFe24 Cell MitoStress Test Kit (Seahorse Bioscience, Massachusetts, USA). A total of 5 × 103 cells per well were seeded in 24-well plates, followed by culturing at 37 °C for 18 h. Growth medium was replaced by medium supplemented with PB and DPB, respectively, and incubated at 37 °C for 36 h. XF assay medium (Seahorse Bioscience) containing glucose (25 mM) and pyruvate (2 mM) or glutamine (2 mM) for OCR and ECAR test, respectively, was added to the wells along with drugs to maintain a stimulating environment. The cells were equilibrated at 37 °C in a CO2-free incubator for 1 h. OCR or ECAR was measured using Seahorse XF24 extracellular flux analyzer, during which oligomycin, FCCP, and rotenone + antimycin A (1 μM each) or glucose (10 mM), oligomycin (1 μM), and 2-DG (130 mM, Sigma−Aldrich) were injected consecutively every 24 min. Data were recorded during the measurement, and the averages of four baseline rates and up to five test rates were used for data analysis. The OCR and ECAR data were normalized to per μg protein. The corresponding ECARs were calculated for glycolysis (before oligomycin injection), glycolytic capacity (after oligomycin injection and deduction of baseline), and glycolytic reserve (after oligomycin injection and deduction of glycolytic capacity). The corresponding OCRs were calculated for basal respiration (OCRinitial − OCRantimycin A/rotenone), ATP production (OCRbasal − OCRoligomycin), max respiration (OCRFCCP − OCRbasal), and spare respiration (OCRFCCP− OCRinitial). MTT assay was basically similar to the neutral red assay except that neutral red dye was replaced by MTT (20 μL, 5 mg mL−1, phosphatebuffered saline) and incubated for 4 h. The final samples were

cell viability(%) = [(ODsample − OD blank )/(ODcontrol − OD blank )] × 100%

Apoptosis was analyzed by fluorescence-activated cell sorting (FACS) using Annexin V and PI staining assay. HeLa cells were seeded in a 6-well plate at a density of 2 × 105 cells per well. The cells were incubated in DMEM (2 mL) and allowed to settle for 24 h. The medium was replaced with the fresh one containing PB, DPB, and cisplatin, respectively. After incubation for 72 h, the cells were washed twice with cold PBS, trypsinized and centrifuged (4000 g, 3 min). The supernatant was discarded, and the cells were resuspended in binding buffer (500 μL), stained with Annexin V, and incubated in the dark for 15 min. The cells were treated with PI and analyzed by flow cytometry. Cellular Uptake. HeLa cells were seeded in a 25 cm2 cultural flask using DMEM supplemented with 10% fetal bovine serum. After incubation for 24 h, the cells were treated with cisplatin, PB, and DPB (1 μM), respectively, for 72 h. The attached cells were washed twice with PBS (4 °C). Cell pellets were collected by centrifugation and then digested with nitric acid (100 μL) at 95 °C for 2 h, followed by the addition of H2O2 (50 μL) and HCl (100 μL) to obtain a fully homogenized solution. The solution was diluted with water, and the Pt content was determined by ICP-MS. The distribution of Pt in cytosol, nucleus, cytoskeleton, and membrane was determined using a FractionPREP cell fractionation kit from BioVision USA. Mitochondria were separated by using a commercially available mitochondria isolation kit (Beyotime Inst. Biotech). DNA Platination and CD Spectroscopy. Genomic DNA of HeLa cells was isolated using a genomic DNA mini preparation kit [Tiangen Biotech (Beijing) Co., LTD]. HeLa cells were seeded in a 6well plate at a density of 2 × 105 cells per well. After incubation at 37 °C for 24 h, the cells were treated with the complex (1 μM) for 48 and 72 h, respectively. The attached cells were washed twice with PBS (4 °C), harvested by trypsinization (0.5 mL), and washed with PBS (1 mL). Cell pallets were lysed in DNAzol reagent (1 mL), and the genomic DNA was extracted from lysate with pure ethanol (0.5 mL) by incubating the sample at room temperature for 1−3 min. The amount of DNA was determined with Nanodrop 1000 at 260 nm, and the Pt level bound to DNA was quantified by ICP-MS. The stock solution of CT-DNA was prepared by dissolving DNA in a buffer solution (5 mM Tris-HCl, 50 mM NaCl, pH 7.4), which was stored in a refrigerator overnight at 4 °C to reach a homogeneous phase and used within 4 days. The concentration of CT-DNA was determined by Nanodrop 1000 at 260 nm, taking 6600 M−1 cm−1 as its absorption coefficient. Samples for CD were prepared by taking a fixed concentration of CT-DNA (60 μM) in the absence and presence of DPB (20 μM) and AsA (40 and 60 μM) and incubated at 37 °C for 48 h in the dark. CD spectra were recorded in the range of 235−320 nm at a scan speed of 10 nm min−1. Cell Cycle. HeLa cells were seeded in a 6-well plate at a density of 2 × 105 cells per well, cultured in DMEM, and allowed to settle for 24 h. The medium was replaced with a fresh one containing cisplatin, PB, and DPB (1 μM), respectively. After incubation for 48 h, the cells H

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Inorganic Chemistry dissolved in DMSO, and the OD value was recorded on an ELISA plate reader at 570 nm. The experiment was done in triplicate.

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(10) Basu, U.; Banik, B.; Wen, R.; Pathak, R. K.; Dhar, S. The platinX series: activation, targeting, and delivery. Dalton Trans. 2016, 45, 12992−13004. (11) Wallace, D. C. Mitochondria and cancer. Nat. Rev. Cancer 2012, 12, 685−698. (12) Ralph, S. J.; Neuzil, J. Mitochondria as targets for cancer therapy. Mol. Nutr. Food Res. 2009, 53, 9−28. (13) Marini, C.; Bianchi, G.; Buschiazzo, A.; Ravera, S.; Martella, R.; Bottoni, G.; Petretto, A.; Emionite, L.; Monteverde, E.; Capitanio, S.; Inglese, E.; Fabbi, M.; Bongioanni, F.; Garaboldi, L.; Bruzzi, P.; Orengo, A. M.; Raffaghello, L.; Sambuceti, G. Divergent targets of glycolysis and oxidative phosphorylation result in additive effects of metformin and starvation in colon and breast cancer. Sci. Rep. 2016, 6, 19569. (14) Papandreou, I.; Goliasova, T.; Denko, N. C. Anticancer drugs that target metabolism: is dichloroacetate the new paradigm. Int. J. Cancer 2011, 128, 1001−1008. (15) Song, H. Q.; Xiao, H. H.; Zhang, Y.; Cai, H. D.; Wang, R.; Zheng, Y. H.; Huang, Y. B.; Li, Y. X.; Xie, Z. G.; Liu, T. J.; Jing, X. B. Multifunctional Pt(IV) pro-drug and its micellar platform: to kill two birds with one stone. J. Mater. Chem. B 2013, 1, 762−772. (16) Zajac, J.; Kostrhunova, H.; Novohradsky, V.; Vrana, O.; Raveendran, R.; Gibson, D.; Kasparkova, J.; Brabec, V. Potentiation of mitochondrial dysfunction in tumor cells by conjugates of metabolic modulator dichloroacetate with a Pt(IV) derivative of oxaliplatin. J. Inorg. Biochem. 2016, 156, 89−97. (17) Dhar, S.; Lippard, S. J. Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22199−22204. (18) Ren, W. X.; Han, J.; Uhm, S.; Jang, Y. J.; Kang, C.; Kim, J. H.; Kim, J. S. Recent development of biotin conjugation in biological imaging, sensing, and target delivery. Chem. Commun. 2015, 51, 10403−10418. (19) Russell-Jones, G.; McTavish, K.; McEwan, J.; Rice, J.; Nowotnik, D. Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours. J. Inorg. Biochem. 2004, 98, 1625−1633. (20) Bildstein, L.; Dubernet, C.; Couvreur, P. Prodrug-based intracellular delivery of anticancer agents. Adv. Drug Delivery Rev. 2011, 63, 3−23. (21) Muhammad, N.; Sadia, N.; Zhu, C.; Luo, C.; Guo, Z. J.; Wang, X. Y. Biotin-tagged platinum(IV) complexes as targeted cytostatic agents against breast cancer cells. Chem. Commun. 2017, 53, 9971− 9974. (22) Choi, Y. W.; Lim, I. K. Sensitization of metformin-cytotoxicity by dichloroacetate via reprogramming glucose metabolism in cancer cells. Cancer Lett. 2014, 346, 300−308. (23) Chen, C. K. J.; Zhang, J. Z.; Aitken, J. B.; Hambley, T. W. Influence of Equatorial and Axial Carboxylato Ligands on the kinetic inertness of platinum(IV) complexes in the presence of ascorbate and cysteine and within DLD-1 cancer cells. J. Med. Chem. 2013, 56, 8757−8764. (24) Leo, A. J. Calculating log Poct from structures. Chem. Rev. 1993, 93, 1281−1306. (25) Maiti, S.; Park, N.; Han, J. H.; Jeon, H. M.; Lee, J. H.; Bhuniya, S.; Kang, C.; Kim, J. S. Gemcitabine-coumarin-biotin conjugates: a target specific theranostic anticancer prodrug. J. Am. Chem. Soc. 2013, 135, 4567−4572. (26) (a) Romero-Canelón, I.; Sadler, P. J. Next-generation metal anticancer complexes: multitargeting via redox modulation. Inorg. Chem. 2013, 52, 12276−12291. (b) Nemirovski, A.; Kasherman, Y.; Tzaraf, Y.; Gibson, D. Reduction of cis trans cis[PtCl2(OCOCH3)2(NH3)2] by aqueous extracts of cancer cells. J. Med. Chem. 2007, 50, 5554−5556. (27) Hall, M. D.; Hambley, T. W. Platinum(IV) antitumour compounds: their bioinorganic chemistry. Coord. Chem. Rev. 2002, 232, 49−67. (28) (a) Zhang, J. Z.; Wexselblatt, E.; Hambley, T. W.; Gibson, D. Pt(IV) analogs of oxaliplatin that do not follow the expected

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.9b00708.

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Synthetic routes; NMR, ESI-MS, HR-ESI-MS, and UV− vis spectra of DPB and PB; assignments of ESI-MS spectra; CD spectra of CT-DNA; flow cytometric analysis, cell cycle, and fluorescence images of HeLa cells; mitochondrial ROS in HeLa cells (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +862589684549. Fax: +862583314502. ORCID

Suxing Jin: 0000-0002-6333-322X Zijian Guo: 0000-0003-4986-9308 Xiaoyong Wang: 0000-0002-8338-9773 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (grants 31570809, 21877059, 31700714), the National Basic Research Program of China (2015CB856300), and Natural Science Foundation of Jiangsu Province (grant BK20150054).

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