The inner mitochondrial membrane fission protein MTP18 serves as a mitophagy receptor to prevent apoptosis in oral cancer.

MTP18 (also known as MTFP1), an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology by regulating mitochondrial fission. Here, we found that MTP18 functions as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with members of the LC3 (also known as MAP1LC3) family through its LC3-interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibited that interaction, thus suppressing mitophagy. Moreover, Parkin or PINK1 deficiency abrogated mitophagy in MTP18-overexpressing human oral cancer-derived FaDu cells. Upon exposure to the mitochondrial oxidative phosphorylation uncoupler CCCP, MTP18[mLIR]-FaDu cells showed decreased TOM20 levels without affecting COX IV levels. Conversely, loss of Parkin or PINK1 resulted in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We also found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. These findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.

Autophagy-modulating phytochemicals in cancer therapeutics: Current evidences and future perspectives.

Autophagy is an intracellular catabolic self-cannibalism that eliminates dysfunctional cytoplasmic cargos by the fusion of cargo-containing autophagosomes with lysosomes to maintain cyto-homeostasis. Autophagy sustains a dynamic interlink between cytoprotective and cytostatic function during malignant transformation in a context-dependent manner. The antioxidant and immunomodulatory phyto-products govern autophagy and autophagy-associated signaling pathways to combat cellular incompetence during malignant transformation. Moreover, in a close cellular signaling circuit, autophagy regulates aberrant epigenetic modulation and inflammation, which limits tumor metastasis. Thus, manipulating autophagy for induction of cell death and associated regulatory phenomena will embark on a new strategy for tumor suppression with wide therapeutic implications. Despite the prodigious availability of lead pharmacophores in nature, the central autophagy regulating entities, their explicit target, as well as pre-clinical and clinical assessment remains a major question to be answered. In addition to this, the stage-specific regulation of autophagy and mode of action with natural products in regulating the key autophagic molecules, control of tumor-specific pathways in relation to modulation of autophagic network specify therapeutic target in caner. Moreover, the molecular pathway specificity and enhanced efficacy of the pre-existing chemotherapeutic agents in co-treatment with these phytochemicals hold high prevalence for target specific cancer therapeutics. Hence, the multi-specific role of phytochemicals in a cellular and tumor context dependent manner raises immense curiosity for investigating of novel therapeutic avenues. In this perspective, this review discusses about diverse implicit mechanisms deployed by the bioactive compounds in diagnosis and therapeutics approach during cancer progression with special insight into autophagic regulation.

Lysosome signaling in cell survival and programmed cell death for cellular homeostasis.

Recent developments in lysosome biology have transformed our view of lysosomes from static garbage disposals that can also act as suicide bags to decidedly dynamic multirole adaptive operators of cellular homeostasis. Lysosome-governed signaling pathways, proteins, and transcription factors equilibrate the rate of catabolism and anabolism (autophagy to lysosomal biogenesis and metabolite pool maintenance) by sensing cellular metabolic status. Lysosomes also interact with other organelles by establishing contact sites through which they exchange cellular contents. Lysosomal function is critically assessed by lysosomal positioning and motility for cellular adaptation. In this setting, mechanistic target of rapamycin kinase (MTOR) is the chief architect of lysosomal signaling to control cellular homeostasis. Notably, lysosomes can orchestrate explicit cell death mechanisms, such as autophagic cell death and lysosomal membrane permeabilization-associated regulated necrotic cell death, to maintain cellular homeostasis. These lines of evidence emphasize that the lysosomes serve as a central signaling hub for cellular homeostasis.

Soybean lectin-triggered IL-6 secretion induces autophagy to kill intracellular mycobacteria through P2RX7 dependent activation of the JAK2/STAT3/Mcl-1 pathway.

Cytokine therapy and cytokine-mediated autophagy have been used as prominent host-directed therapy (HDT) approaches to restrain M. tb growth in the host cell. In the present study, we have dissected the anti-tubercular activity of Soybean lectin (SBL) through cytokine-mediated autophagy induction in differentiated THP-1 (dTHP-1) cells. A significant increase in IL-6 expression was observed in both uninfected and mycobacteria infected dTHP-1 cells through the P2RX7 mediated pathway via PI3K/Akt/CREB-dependent signalling after SBL treatment. Inhibition of IL-6 level using IL-6 neutralizing antibody or associated signalling significantly enhanced the mycobacterial load in SBL-treated dTHP-1 cells. Further, autocrine signalling of IL-6 through its receptor-induced Mcl-1 expression activated autophagy via JAK2/STAT3 pathway, and inhibition of this pathway affected autophagy. Finally, blocking the IL-6-regulated autophagy through NSC 33994 (a JAK2 inhibitor) or S63845 (an Mcl-1 inhibitor) led to a notable increase in intracellular mycobacterial growth in SBL-treated cells. Taken together, these results indicate that SBL interacts with P2RX7 to regulate PI3K/Akt/CREB network to release IL-6 in dTHP-1 cells. The released IL-6, in turn, activates the JAK2/STAT3/Mcl-1 pathway upon interaction with IL-6Rα to modulate autophagy that ultimately controls mycobacterial growth in macrophages.

The emerging, multifaceted role of mitophagy in cancer and cancer therapeutics.

Mitophagy is an evolutionarily conserved cellular process which selectively eliminates dysfunctional mitochondria by targeting them to the autophagosome for degradation. Dysregulated mitophagy results in the accumulation of damaged mitochondria, which plays an important role in carcinogenesis and tumor progression. The role of mitophagy receptors and adaptors including PINK1, Parkin, BNIP3, BNIP3L/NIX, and p62/SQSTM1, and the signaling pathways that govern mitophagy are impaired in cancer. Furthermore, the contribution of mitophagy in regulating the metabolic switch may establish a balance between aerobic glycolysis and oxidative phosphorylation for cancer cell survival. Moreover, ROS-driven mitophagy achieves different goals depending on the stage of tumorigenesis. Mitophagy promotes plasticity in the cancer stem cell through the metabolic reconfiguration for better adaption to the tumor microenvironment. In addition, the present review sheds some light on the role of mitophagy in stemness and differentiation during the transition of cell's fate, which could have a crucial role in cancer progression and metastasis. In conclusion, this review deals with the detailed molecular mechanisms underlying mitophagy, along with highlighting the dual role of mitophagy in different aspects of cancer, suggesting it as a possible target in the mitophagy-modulated cancer therapy.

Epigenetic modifications of autophagy in cancer and cancer therapeutics.

Epigenetic alterations, such as DNA methylation, histone modifications and miRNAs, have a significant role play in malignant cellular transformation and metastasis. On the other hand, autophagy has been reported to perform context-dependent roles in cancer; at times, it becomes lethal and abolishes tumorigenesis, whereas, at other instances, it protects cancer cells by providing a rescue mechanism under adverse conditions. Although epigenetics and autophagy are two important and independent cellular processes, various oncogenic and oncosuppressor proteins involve autophagy through epigenetic modifications and different signaling pathways, thereby regulating tumor growth and therapeutic response. Moreover, the importance of epigenetic modification of autophagy in cancer is reflected through its involvement in cancer stem cell maintenance, which in turn, contributes to tumor cell viability during dormancy leading to tumor recurrence. The effects of epigenetic modifications of autophagy in cancer is still ambiguous and less acknowledged; therefore, efforts have been made to understand its detail underlying mechanism to unveil new targets and avenues for better prognosis and diagnosis of cancer.

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine.

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

Enteromorpha compressa extract induces anticancer activity through apoptosis and autophagy in oral cancer.

Marine algae are an auspicious source of innovative bioactive compounds containing possible therapeutic agents against mammalian cancers. However, the mechanism by which bioactive algal compounds exhibit anticancer activity against oral squamous cell carcinoma (OSCC) is scant. The main objective of the current study was to explore the properties of the Enteromorpha compressa solvent extracts that induced autophagy and apoptosis with reference to their potent phytochemical and antioxidant properties. The presence of bioactive compounds were confirmed by UV and FT-IR spectroscopy. The free radical scavenging activity were analyzed by evaluating H2O2, DPPH, superoxide and hydroxyl activity. The anticancer activities of the extracts were investigated by employing clonogenic and scratch assay. The apoptosis potential was evaluated by DAPI and MMP by Rh123 fluorescence assay. Moreover, the CAT, SOD, GPX, APX, and GR activities were measured. The autophagy potential was evaluated by LC3 puncta formation, acridine orange in addition to LysoTracker staining. The present investigation revealed that the methanolic extract of E. compressa elicited robust free radical scavenging activity that discerns its antiproliferative potency. Moreover, the methanolic algal extract boosted intrinsic apoptosis against OSCC by downregulating protective antioxidant enzymes. Furthermore, it also revealed induction of autophagy to promote cell death in oral cancer cells. The presence of novel bioactive compounds in E. compressa has uncovered possible therapeutic value against OSCC by modulating antioxidant defense system, apoptosis and autophagy that could be used to explore very competent algal candidates for the development of potential alternative anticancer drugs.

Dysregulation of histone deacetylases in carcinogenesis and tumor progression: a possible link to apoptosis and autophagy.

Dysregulation of the epigenome and constitutional epimutation lead to aberrant expression of the genes, which regulate cancer initiation and progression. Histone deacetylases (HDACs), which are highly conserved in yeast to humans, are known to regulate numerous proteins involved in the transcriptional regulation of chromatin structures, apoptosis, autophagy, and mitophagy. In addition, a non-permissive chromatin conformation is created by HDACs, preventing the transcription of the genes encoding the proteins associated with tumorigenesis. Recently, an expanding perspective has been reported from the clinical trials with HDACis (HDAC inhibitors), which has emerged as a determining target for the study of the detailed mechanisms underlying cancer progression. Therefore, the present review focuses on the comprehensive lucubration of post-translational modifications and the molecular mechanisms through which HDACs alter the ambiguities associated with epigenome, with particular insights into the initiation, progression, and regulation of cancer.

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation.

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase-1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi-1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.

N-Doped Carbon Quantum Dot (NCQD)-Deposited Carbon Capsules for Synergistic Fluorescence Imaging and Photothermal Therapy of Oral Cancer.

Use of nanomaterials blessed with both therapeutic and diagnostic properties is a proficient strategy in the treatment of cancer in its early stage. In this context, our paper reports the synthesis of uniform size N-rich mesoporous carbon nanospheres of size 65-70 nm from pyrrole and aniline precursors using Triton-X as a structure-directing agent. Transmission electron microscopy reveals that these carbons spheres contain void spaces in which ultrasmall nitrogen-doped quantum dots (NCQD) are captured within the matrix. These mesoporous hollow NCQD captured carbon spheres (NCQD-HCS) show fluorescence quantum yield up to 14.6% under λex = 340 nm. Interestingly, samples calcined at >800 °C clearly absorb in the wavelength range 700-1000 nm and shows light-to-heat conversion efficiency up to 52%. In vitro experiments in human oral cancer cells (FaDu) show that NCQD-HCS are internalized by the cells and induce a substantial thermal ablation effect in FaDu cells when exposed under a 980 nm near-infrared laser.

Plant lectins in cancer therapeutics: Targeting apoptosis and autophagy-dependent cell death.

Plant lectins are non-immunoglobin in nature and bind to the carbohydrate moiety of the glycoconjugates without altering any of the recognized glycosyl ligands. Plant lectins have found applications as cancer biomarkers for recognizing the malignant tumor cells for the diagnosis and prognosis of cancer. Interestingly, plant lectins contribute to inducing cell death through autophagy and apoptosis, indicating their potential implication in cancer inhibitory mechanism. In the present review, anticancer activities of major plant lectins have been documented, with a detailed focus on the signaling circuit for the possible molecular targeted cancer therapy. In this context, several lectins have exhibited preclinical and clinical significance, driving toward therapeutic potential in cancer treatment. Moreover, several plant lectins induce immunomodulatory activities, and therefore, novel strategies have been established from preclinical and clinical investigations for the development of combinatorial treatment consisting of immunotherapy along with other anticancer therapies. Although the application of plant lectins in cancer is still in very preliminary stage, advanced high-throughput technology could pave the way for the development of lectin-based complimentary medicine for cancer treatment.

Mitochondrial Heterogeneity in Stem Cells.

Mitochondria are customarily acknowledged as the powerhouse of the cell by virtue of their indispensable role in cellular energy production. In addition, it plays an important role in pluripotency, differentiation, and reprogramming. This review describes variation in the stem cells and their mitochondrial heterogeneity. The mitochondrial variation can be described in terms of structure, function, and subcellular distribution. The mitochondria cristae development status and their localization patterns determine the oxygen consumption rate and ATP production which is a central controller of stem cell maintenance and differentiation. Generally, stem cells show spherical, immature mitochondria with perinuclear distribution. Such mitochondria are metabolically less energetic and low polarized. Moreover, mostly glycolytic energy production is found in pluripotent stem cells with a variation in naïve stem cells which perform oxidative phosphorylation (OXPHOS). This article also describes the structural and functional journey of mitochondria during development. Future insight into underlying mechanisms associated with such alternation in mitochondria of stem cells during embryonic stages could uncover mitochondrial adaptability on cellular demands. Moreover, investigating the importance of mitochondria in pluripotency maintenance might unravel the cause of mitochondrial diseases, aging, and regenerative therapies.

Abrus agglutinin stimulates BMP-2-dependent differentiation through autophagic degradation of ß-catenin in colon cancer stem cells.

Eradicating cancer stem cells (CSCs) in colorectal cancer (CRC) through differentiation therapy is a promising approach for cancer treatment. Our retrospective tumor-specimen analysis elucidated alteration in the expression of bone morphogenetic protein 2 (BMP-2) and ß-catenin during the colon cancer progression, indicating that their possible intervention through "forced differentiation" in colon cancer remission. We reveal that Abrus agglutinin (AGG) induces the colon CSCs differentiation, and enhances sensitivity to the anticancer therapeutics. The low dose AGG (max. dose = 100 ng/mL) decreased the expression of stemness-associated molecules such as CD44 and ß-catenin in the HT-29 cell derived colonospheres. Further, AGG augmented colonosphere differentiation, as demonstrated by the enhanced CK20/CK7 expression ratio and induced alkaline phosphatase activity. Interestingly, the AGG-induced expression of BMP-2 and the AGG-induced differentiation were demonstrated to be critically dependent on BMP-2 in the colonospheres. Similarly, autophagy-induction by AGG was associated with colonosphere differentiation and the gene silencing of BMP-2 led to the reduced accumulation of LC3-II, suggesting that AGG-induced autophagy is dependent on BMP-2. Furthermore, hVps34 binds strongly to BMP-2, indicating a possible association of BMP-2 with the process of autophagy. Moreover, the reduction in the self-renewal capacity of the colonospheres was associated with AGG-augmented autophagic degradation of ß-catenin through an interaction with the autophagy adaptor protein p62. In the subcutaneous HT-29 xenograft model, AGG profoundly inhibited the growth of tumors through an increase in BMP-2 expression and LC3-II puncta, and a decrease in ß-catenin expression, confirming the antitumor potential of AGG through induction of differentiation in colorectal cancer.

Mechanism of autophagic regulation in carcinogenesis and cancer therapeutics.

Autophagy in cancer is an intensely debated concept in the field of translational research. The dual nature of autophagy implies that it can potentially modulate the pro-survival and pro-death mechanisms in tumor initiation and progression. There is a prospective molecular relationship between defective autophagy and tumorigenesis that involves the accumulation of damaged mitochondria and protein aggregates, which leads to the production of reactive oxygen species (ROS) and ultimately causes DNA damage that can lead to genomic instability. Moreover, autophagy regulates necrosis and is followed by inflammation, which limits tumor metastasis. On the other hand, autophagy provides a survival advantage to detached, dormant metastatic cells through nutrient fueling by tumor-associated stromal cells. Manipulating autophagy for induction of cell death, inhibition of protective autophagy at tissue-and context-dependent for apoptosis modulation has therapeutic implications. This review presents a comprehensive overview of the present state of knowledge regarding autophagy as a new approach to treat cancer.

Abrus agglutinin promotes irreparable DNA damage by triggering ROS generation followed by ATM-p73 mediated apoptosis in oral squamous cell carcinoma.

Oral cancer, a type of head and neck cancer, is ranked as one of the top most malignancies in India. Herein, we evaluated the anticancer efficacy of Abrus agglutinin (AGG), a plant lectin, in oral squamous cell carcinoma. AGG selectively inhibited cell growth, and caused cell cycle arrest and mitochondrial apoptosis through a reactive oxygen species (ROS)-mediated ATM-p73 dependent pathway in FaDu cells. AGG-induced ROS accumulation was identified as the major mechanism regulating apoptosis, DNA damage and DNA-damage response, which were significantly reversed by ROS scavenger N-acetylcysteine (NAC). Moreover, AGG was found to interact with mitochondrial manganese-dependent superoxide dismutase that might inhibit its activity and increase ROS in FaDu cells. In oral cancer p53 is mutated, thus we focused on p73; AGG resulted in p73 upregulation and knock down of p73 caused a decrease in AGG-induced apoptosis. Interestingly, AGG-dependent p73 expression was found to be regulated by ROS, which was reversed by NAC treatment. A reduction in the level of p73 in AGG-treated shATM cells was found to be associated with a decreased apoptosis. Moreover, administration of AGG (50 µg/kg body weight) significantly inhibited the growth of FaDu xenografts in athymic nude mice. In immunohistochemical analysis, the xenografts from AGG-treated mice displayed a decrease in PCNA expression and an increase in caspase-3 activation as compared to the controls. In conclusion, we established a connection among ROS, ATM and p73 in AGG-induced apoptosis, which might be useful in enhancing the therapeutic targeting of p53 deficient oral squamous cell carcinoma.

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, eIF2α 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. © 2016 Wiley Periodicals, Inc.

Abrus agglutinin targets cancer stem-like cells by eliminating self-renewal capacity accompanied with apoptosis in oral squamous cell carcinoma.

The accumulating evidences show that Abrus agglutinin, a plant lectin, displays a broad range of anticancer activity including cancer-specific induction of apoptosis; however, the underlying molecular mechanism of Abrus agglutinin-induced oral cancer stem cell elimination remains elusive. Our data documented that Abrus agglutinin effectively downregulated the CD44+ expression with the increased CD44- population in different oral cancer cells. After 24-h Abrus agglutinin treatment, FaDu cells were quantified for orosphere formation in ultra-low attachment plates and data showed that Abrus agglutinin inhibited the number and size of orosphere in a dose-dependent manner in FaDu cells. Furthermore, Abrus agglutinin hindered the plasticity of FaDu orospheres as supported by reduced sphere formation and downregulated the self-renewal property via inhibition of Wnt-ß-catenin signaling pathway. Introduction of LiCl, a glycogen synthase kinase 3ß inhibitor, rescued the Abrus agglutinin-stimulated inhibition of ß-catenin and phosphorylated glycogen synthase kinase 3ß in FaDu cell-derived orospheres confirming importance of Wnt signaling in Abrus agglutinin-mediated inhibition of stemness. In this connection, our data showed that Abrus agglutinin restrained proliferation and induced apoptosis in FaDu-derived cancer stem cells in dose-dependent manner. Moreover, western blot data demonstrated that Abrus agglutinin increased the Bax/Bcl-2 ratio with activation of poly(adenosine diphosphate-ribose) polymerase and caspase-3 favoring apoptosis induction in orospheres. Abrus agglutinin induced reactive oxygen species accumulation in orospheres and pretreatment of N-acetyl cysteine, and a reactive oxygen species scavenger inhibited Abrus agglutinin-mediated caspase-3 activity and ß-catenin expression indicating reactive oxygen species as a principal regulator of Wnt signaling and apoptosis. In conclusion, Abrus agglutinin has a potential role as an integrative therapeutic approach for combating oral cancer through targeting self-renewability of orospheres via reactive oxygen species-mediated apoptosis.

Oral Cancer Stem Cells Microenvironment.

Cancer stem cells (CSCs) play important role in tumor growth and metastasis coupled with increased recurrences and acquired therapeutic resistance in oral cancer. The tumor microenvironment imposes intense pressure in cancer evolution in response to adverse growth conditions, resource limitation and immune predation. Here, we discussed the dynamic interplay between cancer stem cells and tumor microenvironment in the formation of intratumoral heterogeneity to modulate tumor progression. The CSCs niche provide a special microhabitat for survival, maintenance of stemness and tumor re-propagation. Moreover, adaptive cellular behavior might be driven by tough tumor microenvironmental selective forces which highly regulate alterations in the gene expression leading to the reprogramming of signaling pathways generating stem-like characteristics, adaptive metabolic plasticity and energy fueling with autophagy to permit the CSCs to sustain in the ever changing microenvironments during tumor progression. On the other hand, CSCs also direct the tumor microenvironment modulation and remodeling in its favour. The cytokines, chemokines and growth factors released from CSCs regulates neoangiogensis, differentiation, degradation of matrix protein and immune suppression favoring tumor-promoting conditions and initiates multiple signaling cascades augmenting the tumor progression.