Shilpi Saha

Curcumin Enhances the Efficacy of Chemotherapy by Tailoring p65NFκB-p300 Cross-talk in Favor of p53-p300 in Breast Cancer

Background: Constitutive activation of NFκB has been found in various cancers, causing resistance to chemotherapeutic drugs. Results: Curcumin pretreatment alleviates p65NFκB activation and hence tailors p65NFκB-p300 cross-talk in favor of p53-p300 in drug-resistant cells. Conclusion: This preclinical study suggests curcumin as a potent chemo-sensitizer to improve the therapeutic index. Significance: These results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug. Breast cancer cells often develop multiple mechanisms of drug resistance during tumor progression, which is the major reason for the failure of breast cancer therapy. High constitutive activation of NFκB has been found in different cancers, creating an environment conducive for chemotherapeutic resistance. Here we report that doxorubicin-induced SMAR1-dependent transcriptional repression and SMAR1-independent degradation of IkBα resulted in nuclear translocation of p65NFκB and its association with p300 histone acetylase and subsequent transcription of Bcl-2 to impart protective response in drug-resistant cells. Consistently SMAR1-silenced drug-resistant cells exhibited IkBα-mediated inhibition of p65NFκB and induction of p53-dependent apoptosis. Interestingly, curcumin pretreatment of drug-resistant cells alleviated SMAR1-mediated p65NFκB activation and hence restored doxorubicin sensitivity. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic death cascade. Importantly, promyelocyte leukemia-mediated SMAR1 sequestration that relieved the repression of apoptosis-inducing genes was indispensable for such chemo-sensitizing ability of curcumin. A simultaneous decrease in drug-induced systemic toxicity by curcumin might also have enhanced the efficacy of doxorubicin by improving the intrinsic defense machineries of the tumor-bearer. Overall, the findings of this preclinical study clearly demonstrate the effectiveness of curcumin to combat doxorubicin-resistance. We, therefore, suggest curcumin as a potent chemo-sensitizer to improve the therapeutic index of this widely used anti-cancer drug. Taken together, these results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug.

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

IntroductionThe 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.MethodsThe 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.ResultsHere, 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.ConclusionsCumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways.

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.

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.

Restoration of tumor suppressor p53 by differentially regulating pro- and anti-p53 networks in HPV-18-infected cervical cancer cells.

Abrogation of functional p53 is responsible for malignant cell transformation and maintenance of human papilloma virus (HPV)-infected cancer cells. Restoration of p53 has, therefore, been regarded as an important strategy for molecular intervention of HPV-associated malignancies. Here we report that differential regulation of pro- and anti-p53 setups not only upregulates p53 transcription but also stabilizes and activates p53 protein to ensure p53-induced apoptosis in HPV-18-infected cervical cancer. Functional restoration of p53 can be achieved by non-steroidal anti-inflammatory drug celecoxib via multiple molecular mechanisms: (i) inhibition of p53 degradation by suppressing viral oncoprotein E6 expression, (ii) promoting p53 transcription by downmodulating cycloxygenase-2 (Cox-2) and simultaneously retrieving p53 from Cox-2 association and (iii) activation of p53 via ataxia telangiectasia mutated-/p38 mitogen-activated protein kinase-mediated phosphorylations at serine-15/-46 residues. That restored p53 is functional has been confirmed by its ability of transactivating Bax and p53-upregulated modulator of apoptosis, which in turn switch on the apoptotic machinery in these cells. Studies undertaken in biopsy samples of cervical carcinoma further validated celecoxib effect. Our approaches involving gene manipulation and pharmacological interference finally highlight that celecoxib alters pro- and anti-p53 networks, not in isolation but in concert, to rejuvenate p53-dependent apoptotic program in HPV-infected cervical cancer 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.

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

Background: HPV18 E6 oncogene represents one of the most promising therapeutic targets for the treatment of HPV-positive tumors. Results: Curcumin-induced SMAR1-HDAC1 recruitment at LCR and E6 region on E6 promoter deacetylates chromatin histones to attenuate c-Fos-mediated E6 transcription to reinstall p53-mediated apoptosis in HPV18-infected cervical cancer. Conclusion: SMAR1 induces E6 repression. Significance: SMAR1 is a repressor of E6-mediated anti-apoptotic network in HPV18-infected cervical cancers. 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.

Non-migratory tumorigenic intrinsic cancer stem cells ensure breast cancer metastasis by generation of CXCR4 + migrating cancer stem cells

Although the role of metastatic cancer stem cells (mCSCs) in tumor progression has been well documented, our study reveals a hitherto unidentified role of tumorigenic intrinsic CSCs (iCSCs) in breast cancer metastasis. We show that unlike highly migratory mCSCs residing in the breast tumor disseminating/peripheral regions, iCSCs populate the inner mass of the tumor and are non-migratory. However iCSCs, via paracrine signaling, induce conversion of non-stem cancer cells to CSCs that (i) are identical to the previously reported mCSCs, and (ii) in contrast to iCSCs, express chemokine receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), which is crucial for their metastatic potential. These mCSCs also demonstrate high in vivo tumorigenicity. Physical non-participation of iCSCs in metastasis is further validated in vivo, where only mCSCs are found to exist in the metastatic sites, lymph nodes and bone marrow, whereas the primary tumor retains both iCSCs and mCSCs. However, iCSCs ensure metastasis since their presence is crucial for deliverance of highly metastatic CXCR4+ mCSCs to the migrating fraction of cells. Cumulatively, these results unveil a novel role of iCSCs in breast cancer metastasis as parental regulators of CXCR4+ mCSCs, and highlight the therapeutic requisite of targeting iCSCs, but not CXCR4+ mCSCs, to restrain breast cancer metastasis from the root by inhibiting the generation of mCSCs from iCSCs. Considering the pivotal role of iCSCs in tumor metastasis, the possibility of metastasis to be a ‘stem cell phenomena’ is suggested.

ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors.

With the existing knowledge of ATM’s role in therapeutic resistance, the present study aimed at identifying the molecular mechanisms that influence ATM to oscillate between chemoresistance and chemosensitivity. We observed that the redox status of tumors functions as a major determinant of ATM-dependent ‘resistance-to-apoptosis’ molecular switch. At a low reactive oxygen species (ROS) condition during genotoxic insult, the ATM/sumoylated-IKKγ interaction induced NFκB activation that resisted JNK-mediated apoptosis, whereas increasing cellular ROS restored ATM/JNK apoptotic signaling. A search for the upstream missing link revealed that high ROS induces oxidation and ubiquitin-mediated degradation of PIASγ, thereby disrupting PIASγ-IKKγ cross talk, a pre-requisite for IKKγ sumoylation and subsequent NFκB activation. Interruption in the PIASγ-mediated resistance pathway channels ATM signaling toward ATM/JNK pro-death circuitry. These in vitro results also translated to sensitive and resistant tumor allograft mouse models in which low ROS-induced resistance was over-ruled in PIASγ knockout tumors, while its overexpression inhibited high ROS-dependent apoptotic cues. Cumulatively, our findings identified an unappreciated yet critical combinatorial function of cellular ROS and PIASγ in regulating ATM-mediated chemosensitization of resistant tumors. Thus, therapeutic strategies employing ROS upregulation to inhibit PIASγ during genotoxic therapy may, in future, help to eliminate the problems of NFκB-mediated tumor drug resistance.

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.