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One of the most promising strategies to increase the efficacy of standard chemotherapy drugs is by combining them with low doses of histone deacetylases inhibitors (HDACis). Regarded as chemosensitizers, the addition of well-tolerated doses of HDACis to platinum-based chemotherapeutics has been proven in vitro and in vivo in recent studies for many cancer types and stages. In this review, we discuss the most commonly used combinations of histone deacetylase inhibitors and platinum based drugs in the context of their possible mechanisms, efficiency, efficacy, and related drawbacks in preclinical and clinical studies.
Cancer can result from defects in genes such as mutations, deletions, and chromosomal abnormalities. These changes may result in the hyper-activation of oncogenes and/or the loss of function of tumor suppressor genes, allowing uncontrolled growth of malignant cells [1,2,3]. Epigenetic modifications, reversibly alter gene expression and can contribute to the initiation and progression of cancer. The reversible nature of epigenetic modifications in contrast to genetic alterations can permit malignant cells to return to a normal state. Recent advancements in the rapidly evolving field of cancer epigenetics show the extensive involvement of epigenetic machinery components in cancer [4,5,6,7,8,9]. Epigenetic abnormalities have been linked not only to cancer, but also to degenerative disorders, neuronal disorders, pediatric syndromes, autoimmune diseases and aging [10,11,12].
Chemotherapy is an often-effective treatment strategy that is designed to kill cancer cells in individuals with certain forms of cancers [13,14]. Many cancer therapies, such as Pt-based drugs, nitrogen-mustards, and drugs like Temozolomide, are based on DNA alkylation [15, 16]. Despite their popularity, the widespread application and efficacy of DNA alkylating agents is hindered by their toxic side effects, their limited activity against many types of cancer, and their susceptibility to acquired drug resistance. Platinum-based drugs such as cisplatin are used for the treatment of many cancers. While cisplatin is known to exert anticancer effects by multiple mechanisms, the best understood mode of action is via the generation of DNA lesions, initiation of the DNA damage response, and induction of mitochondrial apoptosis. Cisplatin treatment very often leads to the development of chemoresistance, thereby causing therapeutic failure. Therefore, an initial therapeutic success, associated with partial response or disease stabilization, is very common for cisplatin monotherapy. Understanding cisplatin resistance mechanism remains a key goal for improving anticancer therapy.
Cisplatin exerts anticancer effects via two intertwined signaling pathways: nuclear and cytoplasmic. Inert cisplatin is intracellularly activated by spontaneous aquation (hydrolysis) reactions in the cytoplasm. The resulting highly reactive molecules interact with a wide variety of cytoplasmic substrates, particularly nucleophiles such as reduced glutathione (GSH), methionine, metallothioneins and proteins. Therefore, cisplatin has the potential to deplete and tilt the redox balance toward oxidative stress and facilitate DNA damage. At the same time, cisplatin is susceptible to inactivation by a number of cytoprotective antioxidant pathways. Aquated cisplatin can also directly bind DNA to generate protein–DNA complexes and DNA–DNA adducts. These DNA-DNA adducts are known to form cisplatin-induced DNA lesions and account for most of the drug’s cytotoxicity. The inactivation of cisplatin by cytoplasmic scavengers limits the amount of reactive cisplatin in the cytoplasm, thus leading to elevated levels of GSH. This phenomenon has been observed in the context of cisplatin resistance, both in vitro and in vivo . Most of the time, the inhibition of single pathways that sustain cisplatin resistance fails to restore sensitivity to normal levels. Effective strategies to both increase efficacy and reduce the adverse side effects of cisplatin chemotherapy treatment are being pursued. Combining classic chemotherapy with epigenetic drugs shows promise as they have been proven to have anti-cancer activity both in vitro and in vivo [18,19,20].
The principal mechanisms by which epigenetic modifications affect tissue-specific gene expression and gene silencing are DNA methylation, chromatin remodeling, microRNA post-transcriptional silencing, and covalent histone modification. Epigenetic changes via covalent modification of DNA and core histones create molecular landmarks that differentially activate or inactivate chromatin. Covalent histone modifications are conserved among eukaryotic organisms and serve as heritable marks for active or inactive chromatin. The regulation of chromosomal structure is mediated in part by modification of the histone core protein amino termini. These covalent modifications which include acetylation, methylation, phosphorylation, and ubiquitination, are a part of normal physiology, but can play a major role in carcinogenesis, with far-reaching implications for human biology and health [21,22]. Drugs that target specific enzymatic processes involved in epigenetic regulation are emerging as effective approaches for the treatment and prevention of certain cancer [23,24,25,26].
Histone acetylation, particularly for histones H3 and H4, is one of the most studied and best understood covalent histone modifications [27,28]. Acetyl groups are transferred to and from lysine residues on the N-terminal tail and on the surface of the nucleosome core. Dynamic regulation of acetylation is facilitated by two enzyme classes: histone acetyltransferases (HAT) and histone deacetylases (HDAC). The opposing actions of HAT and HDAC allow gene expression to be regulated in response to the environment. It has been shown that the changes in transcription due to altered expression levels of HAT and HDAC are closely associated with cancer [29,30]. Reduced levels of histone acetylation as a result of aberrant HDAC activity have been detected in several human tumors and appear to repress tumor suppressor genes, thereby contributing to tumor onset and progression. HDAC inhibitors (HDACis) can reactivate gene expression and inhibit the growth and survival of cancer cells. Several HDACis are currently FDA-approved for the treatment of certain types of cancers, diseases such as bipolar disorder, epilepsy, and depression, but they are finding a multitude of uses in other diseases such as diabetes and HIV [31,32,33]. The specificity of these HDACi compounds for cancer cells and their potency in vitro and in vivo highlight the potential of HDACis as potent agents for the treatment of cancer. HDACis have been shown to sensitize tumor cells to the induction of apoptosis by ionizing radiation, ultraviolet (UV) radiation, and several DNA-damaging agents [34,35,36,37,38].
Over the past several years, targeted cancer drugs have been considered among the most promising developments for obtaining high therapeutic effect with negligible toxicity. Compared to traditional chemotherapy drugs that can affect both healthy and cancer cells, targeted chemotherapy produces fewer adverse effects without impacting efficacy. There are numerous opportunities for targeted therapies because cancer cells utilize multiple intersecting and redundant biochemical pathways to remain viable. Modulating multiple pathways improves upon existing single-target agents, which generally realize only modest improvement and sub-therapeutic effect on solid tumors. Preclinical evidence has shown that adding HDACis to a diverse range of standard chemotherapeutic agents leads to synergistic induction of cancer cell death, demonstrating the utility of a multi-targeted approach involving HDAC modulation in the treatment of cancer. In this review, we focus on HDAC inhibition and platinum-based drug combined therapy for cancer.
Aberrant histone acetylation is observed in a multitude of disorders and cancers, altering gene transcription and ultimately affecting normal cellular behavior. Many malignant conditions demonstrate heightened activity of HAT, exposing transcription factors previously repressed by histone acetylation and altering the association of certain proteins with DNA. Many of the deleterious effects of unregulated HAT activity are mediated by changes in cell cycle control, affecting the function of checkpoint regulators such as p53, E2F, and GATA1. Unregulated HAT activity can also affect chromatin remodeling and promote uncontrolled replication . HDACis are thought to possess anti-cancer activity because of their ability to halt the cell cycle and induce expression of pro-apoptotic proteins that may correct the proliferative state of cancer cells.
While HDAC inhibition alters less than 2% of transcriptional regulation of the entire genome, many important cell cycle regulators become upregulated with HDACis . In a number of cancer cell lines, it was found that the CDKN1A gene encoding p21 was upregulated independently of p53 after treatment with HDAC inhibitors [41,40,42,43]. P21 causes growth arrest, usually in response to injurious stimuli, by inhibiting Cdk2 and halting the cell cycle at G1. Inhibition of Cdk activates retinoblastoma protein (pRb), inhibiting E2F, which is necessary for promotion of the cell cycle to the S phase and preventing further tumor growth. The mechanism of HDACi-induced apoptosis is not currently understood but may involve the re-establishment of tumor suppression machinery such as p53, upregulation of pro-apoptotic proteins, and downregulation of anti-apoptotic signaling . Additionally, the apoptotic effect of HDACis may be drug-specific. Several studies report the activation of the intrinsic (mitochondrial) pathway of apoptosis following administration of SAHA, possibly through a combination of oxidative stress producing reactive oxygen species (ROS) and a reduction in the ability to counter free radicals via downregulation of thioredoxin [45,46]. HDACis may also promote apoptosis by reversing the silencing of pro-apoptosis proteins, including caspase-8, FAS, and APAF1 . In response to the anti-proliferative and pro-apoptotic effects of HDACis, a number of structurally diverse small molecule inhibitors have been developed as cancer therapies [48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63]. In preclinical studies, several classes of HDACis have shown broad and potent anti-tumor activity with low toxicity in normal cells [64,65,61]. Clinically, many HDAC inhibitors have been used as monotherapies for the treatment of cancer (Table 1). PXD101 (Belinostat) has been utilized in clinical trials for hepatocellular carcinoma, multiple myeloma, various ovarian cancers, B-cell non-Hodgkin’s lymphoma, T-cell lymphoma, AML, and several other types of malignant cancers [66,67,68,69,70,71].
The FDA has approved two HDACis for hematological and solid tumors, and several other HDAC-targeted drug candidates are being evaluated clinical trials for other forms of cancer [18,26]. The HDACi suberoylanilide hydroxamic acid (SAHA) was the first HDACi to be approved by the FDA in 2006 for the treatment of cutaneous T-cell lymphoma (CTCL) . The second HDACi a cyclic peptide, romidepsin, received FDA approval in 2009 to treat CTCL in patients who have received at least one prior systemic therapy . While the chemotherapeutic effect of monotherapy with HDAC inhibitor in vitro is obvious, they are known to be effective on only a defined subset of hematological tumors. Their efficacy against solid tumors is significantly less than their efficacy against these hematological tumors; therefore, the combination of HDACis with other cancer drugs is a promising strategy to obtain the full therapeutic potential of these inhibitors in cancer .
Although chemotherapy and HDACis target different sites, the close connection and functional importance of DNA and chromosome structure in cancer development suggests an interaction between these two therapeutic methods [82,83]. The formation of DNA adducts by chemotherapy drugs requires accessibility to the DNA sequence, which can be regulated by altering DNA-associated proteins such as histones. Indeed, HDACis have been regarded as “sensitizer drugs” that display synergistic activity with other agents that alter DNA structure, such as DNA methyltransferase inhibitors and retinoic acids . In addition, the activation of gene expression and induction of apoptosis have been reported in these combinative strategies [84,85].
Although the nature of the molecular mechanisms underpinning combination therapy is largely unknown, higher concentrations of histone deacetylases are believed to exert cell cycle redistribution, induction of apoptosis, and downregulation of survival signals [86,87]. The effect of HDAC inhibition has been attributed at least partly to histone acetylation leading to altered double strand break (DSB) formation and repair. The open chromatin structure induced by histone hyperacetylation renders the DNA more accessible to cellular processes, including damage. Chemotherapeutic agents such as cisplatin bind with DNA to form platinum-DNA adducts; such structural changes may lead to impaired DNA replication and site-specific recombination in cancer cells.
Cisplatin is eliminated from cells by glutathione, the most critical cellular antioxidant. Resistance develops from over-expression of anti-apoptotic proteins Bcl-2 and XIAP, which are associated with increased glutathione synthesis. A proposed mechanism suggests that chromatin relaxation by HDACis may promote accessibility of DNA to transcription factors, thereby downregulating the expression of genes such as Bcl-2 and XIAP and facilitating the formation of platinum-DNA adducts (Fig. 1) . Relaxed chromatin structure and downregulated gene expression help enhance cytotoxicity by giving cisplatin better access to DNA while preventing cells from mitigating the resulting DNA damage. In practice, HDACis (SAHA and Sirtinol in particular) are effective in causing HeLa cells to succumb to the damage caused by platinum-based chemotherapeutics (Fig. 1) .
A number of HDAC inhibitors have been explored in combination with chemotherapeutics, including SAHA, FK-228, MS-275, CI-994, and TSA [89,90,64,82,91,63]. Most of these studies have focused on hematologic cancers, however there are a few that have also examine efficacy in solid tumors . While there are many different treatment combinations of HDACi and Pt drugs for cancer targets awaiting clinical investigation, the drugs most frequently used for combination therapy research with platinum-based chemotherapy are SAHA and valproic acid (VPA).
Cisplatin and various analogues are currently the standard of care for oral squamous cell carcinoma (OSCC). However, cisplatin treatment can result in acquired resistance of cancer cells and dose-dependent systemic toxicity. Many studies have demonstrated the benefit of co-administration of oral SAHA, with cell viability and apoptotic assays showing synergistic induction of cytotoxicity and apoptosis in human tongue, OSCC, Tca8113 and KB cell lines . Furthermore, diverse apoptosis-associated proteins, including p53, BID, cytochrome C and caspase-3, were involved in the induction of apoptosis when treated with cisplatin and subtoxic doses of SAHA. This combinatory treatment is regarded as a potential novel strategy for treatment of OSCC .
Recent studies in OSCC show that combination of cisplatin with SAHA renders 60% of cells apoptotic after 48 hours of treatment and is associated with a number of elevated markers related to endoplasmic reticulum (ER) stress. Caspase-4 and -12 were significantly elevated, with caspase-12-related apoptosis being modulated via the caspase inhibitor z-VAD-fmk [88,94,95,21]. Additionally, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2α), which is often observed in ER stress. This pathway appears to be particularly important in sensitizing cells to cisplatin because this effect is strongly challenged by administration of Salubrinal, a selective eIF-2α phosphatase inhibitor that prevents ER stress-related apoptosis .
The levels of phospho-Akt are reduced in SAHA-treated cells and are associated with increased activity of protein phosphatase 1 (PP1), a stimulator of retinoblastoma protein-mediated apoptosis . These findings indicate that the induction of ER stress and upregulation of its associated pathways are integral to the mechanism by which SAHA enhances apoptosis, particularly by PP1 upregulation and Akt dephosphorylation . Although cisplatin remains a vital mainstay of chemotherapy, the frequency of recurrence and drug resistance leads to poor survival rates for head and neck cancer patients and underscores the need to develop effective modulators of platinating agents . This study has shown that SAHA enhances the efficacy of cisplatin by activating the atypical ER stress pathway to increase apoptosis. Further investigation is required to yield information about the targets of cisplatin and SAHA in this pathway.
Carboplatin is an analog of cisplatin with less nonhematologic toxicity than the parent molecule. A combination of carboplatin and paclitaxel, a non-Pt based mitotic inhibitor, is commonly used to treat solid malignancies, resulting in less platelet toxicity than treatment with carboplatin alone [103,104]. This combination has a toxicity profile that does not overlap with that of SAHA. A phase I clinical study by Ramalingam et al. sought to determine dosing guidelines for SAHA administered on two different schedules with carboplatin and paclitaxel . Further studies on the same combination showed that SAHA improved the efficacy of carboplatin and paclitaxel in patients with advanced non–small-cell lung cancer (NSCLC) [91,63].
Cisplatin is also considered to be an effective tool in chemotherapy for cervical cancer . Severe side effects, such as nephrotoxicity and ototoxicity, limit the dose of cisplatin despite excellent anti-neoplastic activity. In a study performed by Jin et al., a combination treatment of cisplatin and SAHA was tested on HeLa cervical cancer cells . According to this study, low concentrations of SAHA worked synergistically with cisplatin to induce cytotoxicity to a greater extent than with either agent alone. The combination of cisplatin and SAHA induces a marked reduction in cell viability, as determined by Western blot analysis and DAPI staining, showing significantly increased caspase-3 activation and apoptosis in HeLa cells treated with both drugs compared to treatment with cisplatin alone.
Valproic acid (VPA), commonly used to treat epilepsy and bipolar mania, is the second most studied HDACi in combination therapy with platinum-based drugs. It has been reported that the VPA/cisplatin duo amplifies anticancer activity over cisplatin alone in many cancer cell lines, such as head and neck squamous cell carcinoma (HNSCC), human ovarian cancer (SK-OV-3, OVCAR-3, and TOV-21G), and melanoma (M14) [98,97,99].
Vandermeers et al. evaluated the anticancer effect of combining valproate treatment with pemetrexed and cisplatin on mesothelioma cells, a first-line regimen for this malignancy . Present chemotherapeutic regimens are marginally efficacious in malignant pleural mesothelioma, as these tumor cells are particularly resistant to radio- and chemotherapy. Vandermeers utilized cell cultures and xenograft mouse models showing that the inclusion of HDAC inhibitors in the cisplatin and pemetrexed combination may improve the response to treatment. Based on these results, the potential benefit of adding valproate to standard chemotherapy in malignant pleural mesothelioma patients is being evaluated in a phase II clinical trial in refractory and recurrent cases .
HDAC inhibitors MS-275, TSA, and FK-228 have also been shown to enhance activity together with cisplatin on different cancer cell lines [22,100,95,101]. FK228, MS-275 and TSA enhanced the induction of cell death by cisplatin in an ovarian cancer cell line noted for its cisplatin resistance (OVCAR-8). PCl-24781, a highly potent HDAC class I and II inhibitor in various cancer cells at nanomolar concentrations, has shown synergistic anti-cancer effect in soft tissue sarcoma (STS) when combined with low dose conventional chemotherapy. PCI-24781 co-administered with cisplatin has been evaluated in STS cell lines and in vivo mouse studies . Both models show that combined HDAC inhibition and low-dose chemotherapy exhibits anti-STS activity in vitro and in vivo. Increased cancer cell toxicity has been observed when combining cisplatin and TSA in human glioblastoma cell line D54 and breast cancer cell line MCF-7 . The same combination of drugs re-sensitizes human bladder cancer cells that have become resistant to cisplatin . Enhanced cytotoxicity, chemosensitization, and apoptosis are observed in treatment with MS-275 and cisplatin in human oral squamous cell carcinoma .
It is well established that certain cancer cells exhibit a higher level of HDAC expression [105,106,107,108,109,110,111,112]. Furthermore, HDAC inhibitors are known to show preferential selectivity for malignant cells in many cancers. By coupling these effects in a single molecule, it is possible to target multiple pathways simultaneously, as shown in the work by Gleason et al (2008), which combined an HDACi with a nuclear vitamin D receptor ligand . More recently, bifunctional molecules have been designed to improve selectivity by tethering an HDAC inhibitor to a platinum-based DNA binding agent , , , . The HDAC inhibitor, with its known affinity for cancer cells, confers selectivity to the drug and is expected to deliver the chemotherapeutic DNA binding agent alongside the HDAC inhibitor to the tumor , , . These molecules are thought to have a different mechanism from that of classic DNA binding platinum based drugs and are expected to be active against a broader spectrum of human cancer cells compared to classic platinating drugs.
Bifunctional molecules combining HDAC inhibitory and DNA alkylating activity were pioneered by Celine J. Marmion et al. [115,116,117]. As shown in Figure 2, a SAHA derivative was designed to contain a platinum DNA-binding moiety in such a way that its respective actions would ideally remain unchanged. This bifunctional molecule showed marked selectivity for tumor cells relative to non-tumorigenic cells, offering a distinct advantage over treatments involving cisplatin alone. It has been observed that the presence of the malonate linker on the SAHA moiety reduced the anticipated dual functionality once inside the cell nucleus . As such, covalent linker groups should be carefully selected so as not to affect activity. Bifunctional molecules trans-[Pt(VPA–1H)2(NH3)(py)] and trans-[Pt(VPA–1H)2(py)2] containing trans-Pt complexes of the HDACi valproic acid (VPA) were recently reported by the same research group, and treatment with these drugs resulted in apoptosis in A2780 (parental) and A2780 cisR (cisplatin resistant) ovarian cancer cells .
Therapy that induces DNA damage is currently the most popular treatment modality for cancer, manifesting a substantial increase in survival of cancer patients. Unfortunately, some cancers do not respond to the treatment or develop resistance to DNA damaging agents. The utility of HDAC inhibition in chemosensitizers that increase the efficacy of Pt-based chemotherapeutics has been explored in several studies, and HDAC inhibitors were found to benefit even cross-resistant strains of malignancies at very low doses. Preclinical studies carried out in vitro and in vivo by combination therapy with HDACis and standard-of-care platinum-based agents have shown synergistic improvement in cytotoxicity to cancer cells.
Despite evidence showing that HDAC inhibition can enhance anti-cancer efficacy of a diverse range of traditional chemotherapeutics in vitro, the underpinning mechanism remains unverified. By identifying this mechanism of action, it would be possible to rationalize these treatment combinations and optimize them for maximal effect in a variety of cancer types. The effect of any combination regimen using HDACis and platinum drugs can be analyzed by integrating functional cell-based assays with preclinical testing using animal models of cancer to obtain details on mechanisms of action, therapeutic efficacy, and toxicity.
In many cases, issues of selectivity and the emergence of adverse effects have undermined the use of HDACis with classic chemotherapy against many cancers. More recently, novel drugs that complex HDAC-binding agents with platinum-based chemotherapeutics have been synthesized that would ideally reduce the toxic effect these drugs have on normal, healthy cells. Most of these studies are in preclinical phase I testing, awaiting further investigation to evaluate their curative potential. Future work in the field should similarly focus on improving selectivity of these drugs to amplify accumulation in cancer cells at lower, presumably less toxic, doses. Most importantly, understanding the mechanisms by which combination therapy suppresses tumors in each type and stage of cancer is a major challenge that needs to be overcome to advance treatment development.
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