Protective Autophagy in Inducing Stemness and Therapeutic Resistance in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is associated with chemo-radio-resistance due to the existence of a small subset of therapy tolerant intra-tumoral cells called as cancer stem cells (CSCs). Interestingly, we found that stemness surface marker protein CD44, drug resistance marker protein ABCB1, and invasion associated protein ADAM17 are highly co-expressed and their expression is significantly co-related with the tumor progression. This finding implicates the candidature of CD44+ ABCB1+ ADAM17+ cells as a putative CSC compartment in the clinical OSCC specimen and the likeliness of finding a putative stem cell in OSCC is associated with poor differentiation status and high grade of tumors. Further, resistant oral cancer cells were found to have greater stem-like properties when compared to the parental cells as evident from their high sphere forming capacity, possession of higher CD44+ population and increased expression of self-renewal protein β-catenin. The chemoresistant cells had higher autophagic flux and the blockade of autophagy could lead to the down regulation of β-catenin and reduced sphere formation suggesting that autophagy plays a key role in promoting stemness in oral cancer. Interestingly, functional mitophagy was higher in the resistant oral cancer cells suggesting the probable involvement of mitophagy in chemoresistance. Again, we investigated the expression of heat shock chaperone secretory clusterin (sCLU) in OSCC and its association with autophagy. As expected, sCLU is found to be positively correlated with progression of OSCC. Concomitant to the sCLU expression, ATG14 and ULK1 expression increases grade wise in OSCC. Furthermore, our study showed significant interaction of sCLU and ULK1 suggesting the potential role of sCLU in autophagy process. It was shown that the normal human keratinocyte cell HaCaT has low expression of sCLU and LC3-B-II accumulation as compared to the various oral cancer cell lines. Intriguingly, sCLU overexpression could potentially induce autophagy at basal level. Similarly, the expression of mitophagy associated protein PARKIN is positively associated with tumor progression in OSCC. It was unraveled that the stress responder sCLU has a propensity to induce the mitophagy process in the serum starvation condition in oral cancer cells which might have a crucial role in cytoprotection and chemoresistance. Moreover, the meta-analysis study showed that CLU is significantly associated with therapeutic resistance among cancer. Moreover, sCLU-mediated autophagy and/or mitophagy is found to induce cell survival and therapeutic resistance under nutritional and therapeutic stress in oral cancer cells which might have a crucial role in cytoprotection and chemoresistance. In conclusion, this study unravels the role of autophagic and/or mitophagic regulation of acquisition of stem-like properties and chemoresistance in OSCC

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2 phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. (c) 2016 Wiley Periodicals, Inc

Abrus agglutinin is a potent anti-proliferative and anti-angiogenic agent in human breast cancer

Abrus agglutinin (AGG), a plant lectin isolated from the seeds of Abrus precatorius, has documented antitumor and immunostimulatory effects in murine models. To examine possible antitumor activity against breast cancer, we established human breast tumor xenografts in athymic nude mice and intraperitoneally administered AGG. AGG inhibited tumor growth and angiogenesis as confirmed by monitoring the expression of Ki‐67 and CD‐31, respectively. In addition, TUNEL positive cells increased in breast tumors treated with AGG suggesting that AGG mediates anti‐tumorigenic activity through induction of apoptosis and inhibition of angiogenesis. On a molecular level, AGG caused extrinsic apoptosis through ROS generation that was AKT‐dependent in breast cancer cells, without affecting primary mammary epithelial cells, suggesting potential cancer specificity of this natural compound. In addition, using HUVECs, AGG inhibited expression of the pro‐angiogenic factor IGFBP‐2 in an AKT‐dependent manner, reducing angiogenic phenotypes both in vitro and in vivo. Overall, the present results establish that AGG promotes both apoptosis and anti‐angiogenic activities in human breast tumor cells, which might be exploited for treatment of breast and other cancers