Epigenetic reprogramming of T cells: unlocking new avenues for cancer immunotherapy.

T cells, a key component of cancer immunotherapy, undergo a variety of histone modifications and DNA methylation changes since their bone marrow progenitor stages before developing into CD8+ and CD4+ T cells. These T cell types can be categorized into distinct subtypes based on their functionality and properties, such as cytotoxic T cells (Tc), helper T cells (Th), and regulatory T cells (Treg) as subtypes for CD8+ and CD4+ T cells. Among these, the CD4+ CD25+ Tregs potentially contribute to cancer development and progression by lowering T effector (Teff) cell activity under the influence of the tumor microenvironment (TME). This contributes to the development of therapeutic resistance in patients with cancer. Subsequently, these individuals become resistant to monoclonal antibody therapy as well as clinically established immunotherapies. In this review, we delineate the different epigenetic mechanisms in cancer immune response and its involvement in therapeutic resistance. Furthermore, the possibility of epi-immunotherapeutic methods based on histone deacetylase inhibitors and histone methyltransferase inhibitors are under investigation. In this review we highlight EZH2 as the principal driver of cancer cell immunoediting and an immune escape regulator. We have addressed in detail how understanding T cell epigenetic regulation might bring unique inventive strategies to overcome drug resistance and increase the efficacy of cancer immunotherapy.

Lnc-ing epigenetic mechanisms with autophagy and cancer drug resistance.

Long noncoding RNAs (lncRNAs) comprise a diverse class of RNA molecules that regulate various physiological processes and have been reported to be involved in several human pathologies ranging from neurodegenerative disease to cancer. Therapeutic resistance is a major hurdle for cancer treatment. Over the past decade, several studies has emerged on the role of lncRNAs in cancer drug resistance and many trials have been conducted employing them. LncRNAs also regulate different cell death pathways thereby maintaining a fine balance of cell survival and death. Autophagy is a complex cell-killing mechanism that has both cytoprotective and cytotoxic roles. Similarly, autophagy can lead to the induction of both chemosensitization and chemoresistance in cancer cells upon therapeutic intervention. Recently the role of lncRNAs in the regulation of autophagy has also surfaced. Thus, lncRNAs can be used in cancer therapeutics to alleviate the challenges of chemoresistance by targeting the autophagosomal axis. In this chapter, we discuss about the role of lncRNAs in autophagy-mediated cancer drug resistance and its implication in targeted cancer therapy.

Reprogramming Carbohydrate Metabolism in Cancer and Its Role in Regulating the Tumor Microenvironment.

Altered metabolism has become an emerging feature of cancer cells impacting their proliferation and metastatic potential in myriad ways. Proliferating heterogeneous tumor cells are surrounded by other resident or infiltrating cells, along with extracellular matrix proteins, and other secretory factors constituting the tumor microenvironment. The diverse cell types of the tumor microenvironment exhibit different molecular signatures that are regulated at their genetic and epigenetic levels. The cancer cells elicit intricate crosstalks with these supporting cells, exchanging essential metabolites which support their anabolic processes and can promote their survival, proliferation, EMT, angiogenesis, metastasis and even therapeutic resistance. In this context, carbohydrate metabolism ensures constant energy supply being a central axis from which other metabolic and biosynthetic pathways including amino acid and lipid metabolism and pentose phosphate pathway are diverged. In contrast to normal cells, increased glycolytic flux is a distinguishing feature of the highly proliferative cancer cells, which supports them to adapt to a hypoxic environment and also protects them from oxidative stress. Such rewired metabolic properties are often a result of epigenetic alterations in the cancer cells, which are mediated by several factors including, DNA, histone and non-histone protein modifications and non-coding RNAs. Conversely, epigenetic landscapes of the cancer cells are also dictated by their diverse metabolomes. Altogether, this metabolic and epigenetic interplay has immense potential for the development of efficient anti-cancer therapeutic strategies. In this book chapter we emphasize upon the significance of reprogrammed carbohydrate metabolism in regulating the tumor microenvironment and cancer progression, with an aim to explore the different metabolic and epigenetic targets for better cancer treatment.

Suppression of poised oncogenes by ZMYND8 promotes chemo-sensitization.

The major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.

SMAR1 repression by pluripotency factors and consequent chemoresistance in breast cancer stem-like cells is reversed by aspirin.

The high abundance of drug efflux pumps in cancer stem cells (CSCs) contributes to chemotherapy resistance. The transcriptional regulator SMAR1 suppresses CSC expansion in colorectal cancer, and increased abundance of SMAR1 is associated with better prognosis. Here, we found in breast tumors that the expression of SMAR1 was decreased in CSCs through the cooperative interaction of the pluripotency factors Oct4 and Sox2 with the histone deacetylase HDAC1. Overexpressing SMAR1 sensitized CSCs to chemotherapy through SMAR1-dependent recruitment of HDAC2 to the promoter of the gene encoding the drug efflux pump ABCG2. Treating cultured CSCs or 4T1 tumor-bearing mice with the nonsteroidal anti-inflammatory drug aspirin restored SMAR1 expression and ABCG2 repression and enhanced tumor sensitivity to doxorubicin. Our findings reveal transcriptional mechanisms regulating SMAR1 that also regulate cancer stemness and chemoresistance and suggest that, by restoring SMAR1 expression, aspirin might enhance chemotherapeutic efficacy in patients with stem-like tumors.

Regulatory lymphocytes: the dice that resolve the tumor endgame

A large number of cancer patients relapse after chemotherapeutic treatment. The immune system is capable of identifying and destroying cancer cells, so recent studies have highlighted the growing importance of using combinatorial chemotherapy and immunotherapy. However, many patients have innate or acquired resistance to immunotherapies. Long-term follow-up in a pooled meta-analysis exhibited long-term survival in approximately 20% of patients treated with immune checkpoint inhibitors or the adoptive transfer of chimeric T cells. It has been reported that high levels of immunoregulatory cells in cancer patients contribute to immunotherapy resistance via immunosuppression. Among the most important regulatory cell subtypes are the CD4+ T-regulatory cells (Tregs), identified by their expression of the well-characterized, lineage-specific transcription factor FOXP3. In addition to CD4+ Tregs, other regulatory cells present in the tumor microenvironment, namely CD8+ Tregs and IL10-producing B-regulatory cells (Bregs) that also modulate the immune response in solid and lymphoid tumors. These cells together have detrimental effects on tumor immune surveillance and anti-tumor immunity. Therefore, targeting these regulatory lymphocytes will be crucial in improving treatment outcomes for immunotherapy.

Pyridoxine enhances chemo-responsiveness of breast cancer stem cells via redox reconditioning.

A plethora of molecular strategies are employed by breast cancer stem cells (bCSCs) to evade chemotherapy-induced death signals, redox modulation being a crucial factor among those. Here, we observed that bCSCs are resistant to DNA damage and generate low ROS upon doxorubicin (Dox) treatment. Further exploration revealed inherently high NEIL2, a base excision repair (BER) enzyme that plays a key regulatory role in repairing DNA damage, in bCSCs. However, its role in modulating the redox status of bCSCs remains unexplored. In addition, Dox not only upregulates NEIL2 in bCSCs at both transcriptional and translational levels but also declines p300-induced acetylation thus activating NEIL2 and providing a protective effect against the stress inflicted by the genotoxic drug. However, when the redox status of bCSCs is altered by inducing high ROS, apoptosis of the resistant population is accomplished. Subsequently, when NEIL2 is suppressed in bCSCs, chemo-sensitization of the resistant population is enabled by redox reconditioning via impaired DNA repair. This signifies a possibility of therapeutically disrupting the redox balance in bCSCs to enhance their chemo-responsiveness. Our search for an inhibitor of NEIL2 revealed that vitamin B6, i.e., pyridoxine (PN), hinders NEIL2-mediated transcription-coupled repair process by not only decreasing NEIL2 expression but also inhibiting its association with RNA Pol II, thus stimulating DNA damage and triggering ROS. As a consequence of altered redox regulation, bCSCs become susceptible towards Dox, which then induces apoptosis via caspase cascade. These findings signify that PN enhances chemo-responsiveness of bCSCs via redox reconditioning.

A novel role of tumor suppressor ZMYND8 in inducing differentiation of breast cancer cells through its dual-histone binding function.

Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigenetic regulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients, reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancer therapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowed with multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we report another novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, by upregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its antitumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated these findings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiationinducing potential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiation therapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapysensitive. In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate for differentiation therapy in breast cancer.

Molecular targeting of breast and colon cancer cells by PAR1 mediated apoptosis through a novel pro-apoptotic peptide.

A novel activating peptide was designed and synthesized from V. cholerae hemagglutinine protease (HAP) mediated cleavage site of mouse PAR1. The peptide "PFISED" interacts with PAR1 in a new site which is different from its thrombin mediated conventional activation site and induced a series of new downstream signaling pathways. The peptide showed apoptosis in human and mouse breast (MCF-7 and EAC) and colon (HT29 and CT26) cancer cells where as in the same peptide concentration in normal human breast epithelial cells (MCF-10A), normal human fibroblast cells (MRC-5), normal mouse peritoneal macrophage cells and normal mouse breast and colon tissues did not show any effect. Treatment with this peptide enhanced the survival kinetics of EAC induced mice. The peptide mediated apoptosis was inhibited in presence of PAR1 inhibitor and was significantly reduced in si-PAR1 treated cells that indicate the activating peptide "PFISED" induced PAR1 mediated apoptosis of colon and breast cancer cells. This peptide induced over expression and activation of PAR1 and its downstream MAP kinase and NFκB signaling pathways. These signaling pathways enhanced the cellular ROS level to kill malignant cells. We report a novel pro-apoptotic peptide which can selectively kill malignant cells via its specific target receptor PAR1 which is over expressed in the malignant cells and can be used as a molecular target therapy for cancer treatment.

Aspirin Suppresses the Acquisition of Chemoresistance in Breast Cancer by Disrupting an NFκB-IL6 Signaling Axis Responsible for the Generation of Cancer Stem Cells.

Acquired chemoresistance has curtailed cancer survival since the dawn of chemotherapy. Accumulating evidence suggests a major role for cancer stem cells (CSC) in chemoresistance, although their involvement in acquired resistance is still unknown. The use of aspirin has been associated with reduced cancer risk and recurrence, suggesting that the anti-inflammatory drug may exert effects on CSCs. In this study, we investigated the contribution of CSCs to acquired chemoresistance of breast cancer and the avenues for reversing such effects with aspirin. We observed that the residual risk of recurrence was higher in breast cancer patients who had acquired chemoresistance. Treatment of preexisting CSCs with a genotoxic drug combination (5-fluorouracil, doxorubicin, and cyclophosphamide) generated an NFκB-IL6-dependent inflammatory environment that imparted stemness to nonstem cancer cells, induced multidrug resistance, and enhanced the migration potential of CSCs. Treatment with aspirin prior to chemotherapy suppressed the acquisition of chemoresistance by perturbing the nuclear translocation of NFκB in preexisting CSCs. Therefore, disruptions to the NFκB-IL6 feedback loop prevented CSC induction and sensitized preexisting CSCs to chemotherapy. Collectively, our findings suggest that combining aspirin and conventional chemotherapy may offer a new treatment strategy to improve recurrence-free survival of breast cancer patients. Cancer Res; 76(7); 2000-12. ©2016 AACR.

Aspirin inhibits epithelial-to-mesenchymal transition and migration of oncogenic K-ras-expressing non-small cell lung carcinoma cells by down-regulating E-cadherin repressor Slug.

BACKGROUND: Cancer metastasis is one of the most common causes of treatment failure and death in cancer patients. It has been acknowledged that aberrant activation of epithelial-to-mesenchymal transition (EMT) program, endows cancer cells with metastatic competence for which E-cadherin switch is a well-established hallmark. Suppression of E-cadherin by its transcriptional repressor Slug is thus a determining factor for EMT. Here, we aimed at discerning (i) the molecular mechanisms that regulate Slug/E-cadherin axis in oncogenic K-ras-expressing non-small cell lung carcinoma (NSCLC) cells, and (ii) the effect of aspirin in modulating the same. METHODS: The migratory behaviour of NSCLC cell line A549 were deciphered by wound healing assay. Further assessment of the molecular mechanisms was done by western blotting, RT-PCR, confocal microscopy, chromatin immunoprecipitation and small interfering RNA (siRNA)-mediated gene silencing. RESULTS: Here we report that in oncogenic K-ras-expressing A549 cells, Ras/ERK downstream Elk-1 forms p-Elk-1-p300 complex that being directly recruited to SLUG promoter acetylates the same to ensure p65NFκB binding for transcriptional up-regulation of Slug, a transcriptional repressor of E-cadherin. Aspirin inhibits EMT and decelerates the migratory potential of A549 cells by down-regulating Slug and thereby up-regulating E-cadherin. Aspirin impedes activation and nuclear translocation of p65NFκB, essential for this transcription factor being available for SLUG promoter binding. As a consequence, Slug transcription is down-regulated relieving A549 cells from Slug-mediated repression of E-cadherin transcription, thereby diminishing the metastatic potential of these oncogenic Ras-expressing NSCLC cells. CONCLUSIONS: Cumulatively, these results signify a crucial role of the anti-inflammatory agent aspirin as a novel negative regulator of epithelial-to-mesenchymal transition thereby suggesting its candidature as a promising tool for deterring metastasis of highly invasive K-ras-expressing NSCLC cells.

Curcumin and tumor immune-editing: resurrecting the immune system.

Curcumin has long been known to posses medicinal properties and recent scientific studies have shown its efficacy in treating cancer. Curcumin is now considered to be a promising anti-cancer agent and studies continue on its molecular mechanism of action. Curcumin has been shown to act in a multi-faceted manner by targeting the classical hallmarks of cancer like sustained proliferation, evasion of apoptosis, sustained angiogenesis, insensitivity to growth inhibitors, tissue invasion and metastasis etc. However, one of the emerging hallmarks of cancer is the avoidance of immune system by tumors. Growing tumors adopt several strategies to escape immune surveillance and successfully develop in the body. In this review we highlight the recent studies that show that curcumin also targets this process and helps restore the immune activity against cancer. Curcumin mediates several processes like restoration of CD4(+)/CD8(+) T cell populations, reversal of type-2 cytokine bias, reduction of Treg cell population and suppression of T cell apoptosis; all these help to resurrect tumor immune surveillance that leads to tumor regression. Thus interaction of curcumin with the immune system is also an important feature of its multi-faceted modes of action against cancer. Finally, we also point out the drawbacks of and difficulties in curcumin administration and indicate the use of nano-formulations of curcumin for better therapeutic efficacy.

Sulphur alters NFκB-p300 cross-talk in favour of p53-p300 to induce apoptosis in non-small cell lung carcinoma.

Adverse side effects of chemotherapy during cancer treatment have shifted considerable focus towards therapies that are not only targeted but are also devoid of toxic side effects. We evaluated the antitumorigenic activity of sulphur, and delineated the molecular mechanisms underlying sulphur-induced apoptosis in non-small cell lung carcinoma (NSCLC) cells. A search for the underlying mechanism revealed that the choice between the two cellular processes, NFκBp65-mediated survival and p53-mediated apoptosis, was decided by the competition for a limited pool of transcriptional coactivator protein p300 in NSCLC cells. In contrast, sulphur inhibited otherwise upregulated survival signaling in NSCLC cells by perturbing the nuclear translocation of p65NFκB, its association with p300 histone acetylase, and subsequent transcription of Bcl-2. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic mitochondrial death cascade. Overall, the findings of this preclinical study clearly delineated the molecular mechanism underlying the apoptogenic effect of the non-toxic homeopathic remedy, sulphur, in NSCLC cells.

Mithramycin A sensitizes therapy-resistant breast cancer stem cells toward genotoxic drug doxorubicin.

Chemotherapy resistance is a major clinical challenge for the management of locally advanced breast cancer. Accumulating evidence suggests a major role of cancer stem cells (CSCs) in chemoresistance evoking the requirement of drugs that selectively target CSCs in combination with chemotherapy. Here, we report that mithramycin A, a known specificity protein (Sp)1 inhibitor, sensitizes breast CSCs (bCSCs) by perturbing the expression of drug efflux transporters, ATP-binding cassette sub-family G, member 2 (ABCG2) and ATP-binding cassette sub-family C, member 1 (ABCC1), survival factors, B-cell lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP), and, stemness regulators, octamer-binding transcription factor 4 (Oct4) and Nanog, which are inherently upregulated in these cells compared with the rest of the tumor population. In-depth analysis revealed that aberrant overexpression of Sp1 in bCSCs transcriptionally upregulates (1) resistance-promoting genes to protect these cells from genotoxic therapy, and (2) stemness regulators to sustain self-renewal potential of these cells. However, mithramycin A causes transcriptional suppression of these chemoresistant and self-renewal genes by inhibiting Sp1 recruitment to their promoters. Under such antisurvival microenvironment, chemotherapeutic agent doxorubicin induces apoptosis in bCSCs via DNA damage-induced reactive oxygen species generation. Cumulatively, our findings raise the possibility that mithramycin A might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to eliminate CSCs. This will consequently lead to the improvement of therapeutic outcome for the treatment-resistant breast carcinomas.

Curcumin inhibits breast cancer stem cell migration by amplifying the E-cadherin/ß-catenin negative feedback loop.

INTRODUCTION: The existence of cancer stem cells (CSCs) has been associated with tumor initiation, therapy resistance, tumor relapse, angiogenesis, and metastasis. Curcumin, a plant ployphenol, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating (i) the mechanisms underlying the aggravated migration potential of breast CSCs (bCSCs) and (ii) the effects of curcumin in modulating the same. METHODS: The migratory behavior of MCF-7 bCSCs was assessed by using cell adhesion, spreading, transwell migration, and three-dimensional invasion assays. Stem cell characteristics were studied by using flow cytometry. The effects of curcumin on bCSCs were deciphered by cell viability assay, Western blotting, confocal microscopy, and small interfering RNA (siRNA)-mediated gene silencing. Evaluations of samples of patients with breast cancer were performed by using immunohistochemistry and flow cytometry. RESULTS: Here, we report that bCSCs are endowed with aggravated migration property due to the inherent suppression of the tumor suppressor, E-cadherin, which is restored by curcumin. A search for the underlying mechanism revealed that, in bCSCs, higher nuclear translocation of beta-catenin (i) decreases E-cadherin/beta-catenin complex formation and membrane retention of beta-catenin, (ii) upregulates the expression of its epithelial-mesenchymal transition (EMT)-promoting target genes (including Slug), and thereby (iii) downregulates E-cadherin transcription to subsequently promote EMT and migration of these bCSCs. In contrast, curcumin inhibits beta-catenin nuclear translocation, thus impeding trans-activation of Slug. As a consequence, E-cadherin expression is restored, thereby increasing E-cadherin/beta-catenin complex formation and cytosolic retention of more beta-catenin to finally suppress EMT and migration of bCSCs. CONCLUSIONS: Cumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.

Inhibition of epithelial to mesenchymal transition by E-cadherin up-regulation via repression of slug transcription and inhibition of E-cadherin degradation: dual role of scaffold/matrix attachment region-binding protein 1 (SMAR1) in breast cancer cells.

The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor.

Nuclear matrix protein SMAR1 represses c-Fos-mediated HPV18 E6 transcription through alteration of chromatin histone deacetylation.

Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.

Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to restrain angiogenesis.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.

Contribution of the ROS-p53 feedback loop in thuja-induced apoptosis of mammary epithelial carcinoma cells.

The adverse side-effects associated with chemotherapy during cancer treatment have shifted considerable focus towards therapies that are targeted but devoid of toxic side-effects. In the present study, the antitumorigenic activity of thuja, the bioactive derivative of the medicinal plant Thuja occidentalis, was evaluated, and the molecular mechanisms underlying thuja-induced apoptosis of functional p53-expressing mammary epithelial carcinoma cells were elucidated. Our results showed that thuja successfully induced apoptosis in functional p53-expressing mammary epithelial carcinoma cells. Abrogation of intracellular reactive oxygen species (ROS), prevention of p53-activation, knockdown of p53 or inhibition of its functional activity significantly abridged ROS generation. Notably, under these conditions, thuja-induced breast cancer cell apoptosis was reduced, thereby validating the existence of an ROS-p53 feedback loop. Elucidating this feedback loop revealed bi-phasic ROS generation as a key mediator of thuja-induced apoptosis. the first phase of ROS was instrumental in ensuring activation of p53 via p38MAPK and its nuclear translocation for transactivation of Bax, which induced a second phase of mitochondrial ROS to construct the ROS-p53 feedback loop. Such molecular crosstalk induced mitochondrial changes i) to maintain and amplify the thuja signal in a positive self-regulatory feedback manner; and ii) to promote the mitochondrial death cascade through cytochrome c release and caspase-driven apoptosis. These results open the horizon for developing a targeted therapy by modulating the redox status of functional p53-expressing mammary epithelial carcinoma cells by thuja.

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells.

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.