Fayaz Malik

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by Expanding the Cancer Stem Cell Population

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine.

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψmt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.

HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy of Adjuvant Trastuzumab

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of Th1 immunostimulatory chemical entity.

Withania somnifera, commonly called Ashwagandha in the Indian traditional system of medicine has been reported for several pharmacological activities. This study demonstrates, for the first time, the potential role of the chemically standardized leaf extract of W. somnifera (WSL) and its identified component in activating immune system. WSL enhanced Th1 cytokine IFN-gamma expression in Con A primed splenocytes in vitro. When given orally for 2 weeks to BALB/c mice immunized with emulsion of OVA in Freunds adjuvant (OVA-FCA), it caused dose-dependent proliferation of T cells and improved their ability to secrete IL-2 and IFN-gamma, but moderately down-regulated Th2 cytokine IL-4. Flow cytometric analysis of lymphocyte surface markers of T cells CD3(+), CD4(+) and CD8(+), and B cells CD19(+) indicated prominent enhancement in proliferation and differentiation of lymphocytes. Further, the effect of WSL in immunized mice elicited up-regulation of beta-integrins LFA (CD11a) and Mac-1 (CD11b) in splenocytes. Co-stimulatory molecules CD80 and CD86 that are important secondary signals for the activation of immune system elicited remarkable enhanced expression when observed in spleen-derived macrophages isolated from WSL treated mice. Chemical standardization of WSL suggested that the withanolide 2,3 dihydro-3-sulphonile withanone is a major constituent of WSL responsible for skewing to Th1 immune polarization by stimulating the expression of IFN-gamma and B cell switch over to secrete IgG2a while simultaneously enhancing the expression of co-stimulatory molecules and integrins. These studies demonstrate the possible usefulness of WSL and its major constituent WSL-2 as Th1 immune adjuvants for chronic infectious ailments where patients suffer from weakened Th1 immunity.

Activation of p53/p21/PUMA alliance and disruption of PI-3/Akt in multimodal targeting of apoptotic signaling cascades in cervical cancer cells by a pentacyclic triterpenediol from Boswellia serrata.

Cervical carcinoma is a growing menace to women health worldwide. This study reports the apoptotic cell death in human cervical cancer HeLa and SiHa cells by a pentacyclic triterpenediol (TPD) from Boswellia serrata by a mechanism different from reported in HL‐60 cells. It caused oxidative stress by early generation of nitric oxide and reactive oxygen species that robustly up regulated time‐dependent expression of p53/p21/PUMA while conversely abrogating phosphatidylinositol‐3‐kinase (PI3K)/Akt pathways in parallel. TPD also decreased the expression of PI3K/pAkt, ERK1/2, NF‐κB/Akt signaling cascades which coordinately contribute to cancer cell survival through these distinct pathways. The tumor suppressor p53 pathway predominantly activated by TPD further up‐regulated PUMA, which concomitantly decreased the Bcl‐2 level, caused mitochondrial membrane potential loss with attendant translocation of Bax and drp1 to mitochondria and release of pro‐apoptotic factors such as cytochrome c and Smac/Diablo to cytosol leading to caspases‐3 and ‐9 activation. In addition both the phospho‐p53 and p21 were found to accumulate heavily in the nuclear fraction with attendant decrease in topoisomarase II and survivin levels. On the contrary, TPD did not affect the extrinsic signaling transduction pathway effectively through apical death receptors. Interestingly, N‐acetyl cysteine, ascorbate and s‐methylisothiourea (sMIT) rescued cells significantly from TPD induced DNA damage and caspases activation. TPD may thus find usefulness in managing and treating cervical cancer.

Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Monarda citriodora.

We have isolated an essential oil from Monarda citriodora (MC) and characterized its 22 chemical constituents with thymol (82%), carvacrol (4.82%), β-myrcene (3.45%), terpinen-4-ol (2.78%) and p-cymene (1.53%) representing the major constituents. We have reported for the first time the chemotherapeutic potential of MC in human promyelocytic leukemia HL-60 cells by means of apoptosis and disruption of the PI3K/AKT/mTOR signaling cascade. MC and its major constituent, thymol, inhibit the cell proliferation in different types of cancer cell lines like HL-60, MCF-7, PC-3, A-549 and MDAMB-231. MC was found to be more cytotoxic than thymol in HL-60 cells with an IC50 value of 22 μg/ml versus 45 μg/ml for thymol. Both MC and thymol induce apoptosis in HL-60 cells, which is evident by Hoechst staining, cell cycle analysis and immuno-expression of Bcl-xL, caspase-3,-8,-9 and PARP-1 cleavage. Both induce apoptosis by extrinsic and intrinsic apoptotic pathways that were confirmed by enhanced expression of death receptors (TNF-R1, Fas), caspase-9, loss of mitochondrial membrane potential and regression of Bcl-2/Bax ratio. Interestingly, both MC and thymol inhibit the downstream and upstream signaling of PI3K/AKT/mTOR pathway. The degree of apoptosis induction and disruption of the PI3K signaling cascade by MC was significantly higher when compared to thymol.

Secalonic Acid-D Represses HIF1α/VEGF-Mediated Angiogenesis by Regulating the Akt/mTOR/p70S6K Signaling Cascade

Tumor angiogenesis is a validated target for therapeutic intervention, but agents that are more disease selective are needed. Here, we report the isolation of secalonic acid-D (SAD), a mycotoxin from a novel source that exhibits potent antiangiogenic antitumor activity. SAD inhibited multiple HIF1α/VEGF-arbitrated angiogenesis dynamics as scored in human umbilical vascular endothelial cells and human MCF-7 breast tumor xenografts. Similarly, SAD suppressed VEGF-induced microvessel sprouting from rat aortic ring and blood vessel formation in the Matrigel plug assay in C57/BL6J mice. Under normoxic or hypoxic conditions, SAD inhibited cell survival through the Akt/mTOR/p70S6K pathway, with attendant effects on key proangiogenesis factors, including HIF1α, VEGFR, and MMP-2/MMP-9. These effects were reversed by cotreatment with the Akt inhibitors perifosine and GSK69069 or by the addition of neutralizing VEGF antibodies. The apoptotic properties of SAD were determined to be both extrinsic and intrinsic in nature, whereas the cell-cycle inhibitory effects were mediated by altering the level of key G1-S transition-phase proteins. In experimental mouse models of breast cancer, SAD dosing produced no apparent toxicities (either orally or intraperitoneal) at levels that yielded antitumor effects. Taken together, our findings offered a preclinical validation and mechanistic definition of the antiangiogenic activity of a novel mycotoxin, with potential application as a cancer-selective therapeutic agent.

Immunomodulatory activity of biopolymeric fraction BOS 2000 from Boswellia serrata

Oral administration of BOS 2000 (1–10 mg/kg) elicited a dose related increase in the delayed hypersensitivity reaction (early 24 h and delayed 48 h) in mice. It also stimulated the IgM and IgG titre expressed in the form of plaques (PFC) and complement fixing antibody titre. The concentration of cytokines (IL‐4, IFN‐γ and TNF‐α) in serum with respect to T cell interactions, i.e. (CD4/CD8) and the proliferation of lymphocytes were significantly increased at 10 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of BOS 2000 in a dose‐dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF‐α and IFN‐γ production as a mode of action. Copyright