SIRT1-activating butein inhibits arecoline-induced mitochondrial dysfunction through PGC1α and MTP18 in oral cancer.

BACKGROUND: Mitochondrial dysfunction associated with mitochondrial DNA mutations, enzyme defects, generation of ROS, and altered oxidative homeostasis is known to induce oral carcinogenesis during exposure to arecoline. Butein, a natural small molecule from Butea monosperma, possesses anti-inflammatory, anti-diabetic, and anti-cancer effects. However, the role of butein in the mitochondrial quality control mechanism has not been illuminated clearly. PURPOSE: This study aimed to explore the role of butein in preserving mitochondrial quality control during arecoline-induced mitochondrial dysfunction in oral cancer to curtail the early onset of carcinogenesis. METHODS: Cell viability was evaluated by MTT assay. The relative protein expressions were determined by western blotting. Immunofluorescence and confocal imaging were used to analyze the relative fluorescence and co-localization of proteins. Respective siRNAs were used to examine the knockdown-based studies. RESULTS: Butein, in the presence of arecoline, significantly caused a decrease in mitochondrial hyperpolarization and ROS levels in oral cancer cells. Mechanistically, we found an increase in COXIV, TOM20, and PGC1α expression during butein treatment, and inhibition of PGC1α blunted mitochondrial biogenesis and decreased the mitochondrial pool. Moreover, the fission protein MTP18, and its molecular partners DRP1 and MFF were dose-dependently increased during butein treatment to maintain mitochondria mass. In addition, we also found increased expression of various mitophagy proteins, including PINK1, Parkin, and LC3 during butein treatment, suggesting the clearance of damaged mitochondria to maintain a healthy mitochondrial pool. Interestingly, butein increased the activity of SIRT1 to enhance the functional mitochondrial pool, and inhibition of SIRT1 found to reduce the mitochondrial levels, as evident from the decrease in the expression of PGC1α and MTP18 in oral cancer cells. CONCLUSION: Our study proved that SIRT1 maintains a functional mitochondrial pool through PGC1α and MTP18 for biogenesis and fission of mitochondria during arecoline exposure and could decrease the risk of mitochondria dysfunctionality associated with the onset of oral carcinogenesis.

Recent advancement of autophagy in polyploid giant cancer cells and its interconnection with senescence and stemness for therapeutic opportunities.

Recurrent chemotherapy-induced senescence and resistance are attributed to the polyploidization of cancer cells that involve genomic instability and poor prognosis due to their unique form of cellular plasticity. Autophagy, a pre-dominant cell survival mechanism, is crucial during carcinogenesis and chemotherapeutic stress, favouring polyploidization. The selective autophagic degradation of essential proteins associated with cell cycle progression checkpoints deregulate mitosis fidelity and genomic integrity, imparting polyploidization of cancer cells. In connection with cytokinesis failure and endoreduplication, autophagy promotes the formation, maintenance, and generation of the progeny of polyploid giant cancer cells. The polyploid cancer cells embark on autophagy-guarded elevation in the expression of stem cell markers, along with triggered epithelial and mesenchymal transition and senescence. The senescent polyploid escapers represent a high autophagic index than the polyploid progeny, suggesting regaining autophagy induction and subsequent autophagic degradation, which is essential for escaping from senescence/polyploidy, leading to a higher proliferative phenotypic progeny. This review documents the various causes of polyploidy and its consequences in cancer with relevance to autophagy modulation and its targeting for therapeutic intervention as a novel therapeutic strategy for personalized and precision medicine.

Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma.

BACKGROUND: Bacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. AIM: The aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. RESULTS: We performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice. CONCLUSION: In conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Stonin 2 activates lysosomal-mTOR axis for cell survival in oral cancer.

Aberrant expression of various genes is associated with the progression of oral squamous cell carcinoma. Stonin 2, an endocytic protein, has a prominent role in clathrin-associated endocytosis. Its position in oral cancer is still unknown. Here, we report that STON2 expression increases with an increase in the grade of the oral cancer tissue. Further, STON2 overexpressed cells possess a higher rate of proliferation and migraton in oral cancer cells. STON2 helps maintain lysosomal functions by preserving the lysosomal membrane integrity. It activates the Akt-mTOR axis and retains the mTOR on the membrane of the lysosomes. Further, we have identified an inhibitor of STON2, i.e., Trifluoperazine dihydrochloride (TFP), which targets the lysosomal axis by disrupting the Akt-mTOR pathway and causes lysosomal membrane permeabilization. Intererstingly, TFP shows a decrease in cell vaibility on the oral cancer cells and it was observed that cell viability is restored in TFP-treated STON2 overexpressed cells. Moreover, the lysosomal activity and the Akt-mTOR expression are restored in STON2 overexpressed cells co-treated with TFP, establishing TFP targets STON2 to showcase its anti-cancer effects in oral cancer. In conclusion, STON2 might serve as a potential biomarker in oral cancer, and its inhibition could functions as a novel anti-cancer mechanims against oral cancer.

Autophagy for secretory protein: Therapeutic targets in cancer.

Autophagy, a classical cellular degradative catabolic process, also involves a functionally discrete non-degradative role in eukaryotic cells. It imparts critical regulatory function on conventional and unconventional protein secretion (degradative and secretory autophagy with distinct lysosomal degradation and extracellular expulsion, respectively) pathways. The N-amino terminal leader sequence containing proteins follows a conventional secretion pathway, while the leader-less proteins opt for secretory autophagy. The secretory autophagic process ensembles core autophagy machinery proteins, specifically ULK1/2, Beclin 1, LC3, and GABARAP, in coordination with Golgi re-assembly and stacking proteins (GRASPs). The secretory omegasomes fuse with the plasma membrane for the expulsion of cytosolic cargos to the extracellular environment. Alternatively, the secretory omegasomes also fuse with multi-vesicular bodies (MVBs) and harmonize ESCRTs (Complex I; TSG101) and Rab GTPase for their release to extracellular space. Autophagy has been associated with the secretion of diverse proteins involved in cellular signaling, inflammation, and carcinogenesis. Secreted proteins play an essential role in cancer by sustaining cell proliferation, inhibiting apoptosis, enhancing angiogenesis and metastasis, immune cell regulation, modulation of cellular energy metabolism, and resistance to anticancer drugs. The complexity of autophagy regulation during tumorigenesis is dependent on protein secretion pathways. Autophagy-regulated TOR-autophagy spatial coupling compartment complex energizes enhanced secretion of pro-inflammatory cytokines and leaderless proteins such as HMGB1. In conclusion, the chapter reviews the role of autophagy in regulating conventional and unconventional protein secretion pathways and its possible role in cancer.

Recent progress of autophagy signaling in tumor microenvironment and its targeting for possible cancer therapeutics.

Autophagy, a lysosomal catabolic process, involves degradation of cellular materials, protein aggregate, and dysfunctional organelles to maintain cellular homeostasis. Strikingly, autophagy exhibits a dual-sided role in cancer; on the one hand, it promotes clearance of transformed cells and inhibits tumorigenesis, while cytoprotective autophagy has a role in sustaining cancer. The autophagy signaling in the tumor microenvironment (TME) during cancer growth and therapy is not adequately understood. The review highlights the role of autophagy signaling pathways to support cancer growth and progression in adaptation to the oxidative and hypoxic context of TME. Furthermore, autophagy contributes to regulating the metabolic switch for generating sufficient levels of high-energy metabolites, including amino acids, ketones, glutamine, and free fatty acids for cancer cell survival. Interestingly, autophagy has a critical role in modulating the tumor-associated fibroblast resulting in different cytokines and paracrine signaling mediated angiogenesis and invasion of pre-metastatic niches to secondary tumor sites. Moreover, autophagy promotes immune evasion to inhibit antitumor immunity, and autophagy inhibitors enhance response to immunotherapy with infiltration of immune cells to the TME niche. Furthermore, autophagy in TME maintains and supports the survival of cancer stem cells resulting in chemoresistance and therapy recurrence. Presently, drug repurposing has enabled the use of lysosomal inhibitor-based antimalarial drugs like chloroquine and hydroxychloroquine as clinically available autophagy inhibitors in cancer therapy. We focus on the recent developments of multiple autophagy modulators from pre-clinical trials and the challenges in developing autophagy-based cancer therapy.

Inflammasomes in cancer: Effect of epigenetic and autophagic modulations.

Tumour-promoting inflammation is a critical hallmark in cancer development, and inflammasomes are well-known regulators of inflammatory processes within the tumour microenvironment. Different inflammasome components along with the adaptor, apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC), and the effector, caspase-1, have a significant influence on tumorigenesis but in a tissue-specific and stage-dependent manner. The downstream products of inflammasome activation, that is the proinflammatory cytokines such as IL-1ß and IL-18, regulate tissue homeostasis and induce antitumour immune responses, but in contrast, they can also favour cancer growth and proliferation by directing various oncogenic signalling pathways in cancer cells. Moreover, different epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNAs, control inflammasomes and their components by regulating gene expression during cancer progression. Furthermore, autophagy, a master controller of cellular homeostasis, targets inflammasome-induced carcinogenesis by maintaining cellular homeostasis and removing potential cancer risk factors that promote inflammasome activation in support of tumorigenesis. Here, in this review, we summarize the effect of inflammasome activation in cancers and discuss the role of epigenetic and autophagic regulatory mechanisms in controlling inflammasomes. A proper understanding of the interactions among these key processes will be useful for developing novel therapeutic regimens for targeting inflammasomes in cancer.

The lysosome as an imperative regulator of autophagy and cell death.

Lysosomes are single membrane-bound organelles containing acid hydrolases responsible for the degradation of cellular cargo and maintenance of cellular homeostasis. Lysosomes could originate from pre-existing endolysosomes or autolysosomes, acting as a critical juncture between autophagy and endocytosis. Stress that triggers lysosomal membrane permeabilization can be altered by ESCRT complexes; however, irreparable damage to the membrane results in the induction of a selective lysosomal degradation pathway, specifically lysophagy. Lysosomes play an indispensable role in different types of autophagy, including microautophagy, macroautophagy, and chaperone-mediated autophagy, and various cell death pathways such as lysosomal cell death, apoptotic cell death, and autophagic cell death. In this review, we discuss lysosomal reformation, maintenance, and degradation pathways following the involvement of the lysosome in autophagy and cell death, which are related to several pathophysiological conditions observed in humans.

Mitochondrial dysfunction as a driver of NLRP3 inflammasome activation and its modulation through mitophagy for potential therapeutics.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is responsible for the sensation of various pathogenic and non-pathogenic damage signals and has a vital role in neuroinflammation and neural diseases. Various stimuli, such as microbial infection, misfolded protein aggregates, and aberrant deposition of proteins can induce NLRP3 inflammasome in neural cells. Once triggered, the NLRP3 inflammasome leads to the activation of caspase-1, which in turn activates inflammatory cytokines, such as interleukin-1ß and interleukin -18, and induces pyroptotic cell death. Mitochondria are critically involved in diverse cellular processes and are involved in regulating cellular redox status, calcium levels, inflammasome activation, and cell death. Mitochondrial dysfunction and subsequent accumulation of mitochondrial reactive oxygen species, mitochondrial deoxyribonucleic acid, and other mitochondria-associated proteins and lipids play vital roles in the instigation of the NLRP3 inflammasome. In addition, the processes of mitochondrial dynamics, such as fission and fusion, are essential in the maintenance of mitochondrial integrity and their imbalance also promotes NLRP3 inflammasome activation. In this connection, mitophagy-mediated maintenance of mitochondrial homeostasis restricts NLRP3 inflammasome hyperactivation and its consequences in various neurological disorders. Hence, mitophagy can be exploited as a potential strategy to target damaged mitochondria induced NLRP3 inflammasome activation and its lethal consequences. Therefore, the identification of novel mitophagy modulators has promising therapeutic potential for NLRP3 inflammasome-associated neuronal diseases.

Intricate role of mitochondrial calcium signalling in mitochondrial quality control for regulation of cancer cell fate.

Mitochondrial quality control is crucial for sustaining cellular maintenance. Mitochondrial Ca2+ plays an important role in the maintenance of mitochondrial quality control through regulation of mitochondrial dynamics, mitophagy and mitochondrial biogenesis for preserving cellular homeostasis. The regulation of this dynamic interlink between these mitochondrial networks and mitochondrial Ca2+ appears indispensable for the adaptation of cells under external stimuli. Moreover, dysregulation of mitochondrial Ca2+ divulges impaired mitochondrial control that results in several pathological conditions such as cancer. Hence this review untangles the interplay between mitochondrial Ca2+ and quality control that govern mitochondrial health and mitochondrial coordinates in the development of cancer.

Mitochondrial rewiring through mitophagy and mitochondrial biogenesis in cancer stem cells: A potential target for anti-CSC cancer therapy.

Cancer stem cells (CSCs) are distinct subpopulations of cancer cells with stem cell-like abilities and are more resilient to chemotherapy, causing tumor relapse. Mitophagy, a selective form of autophagy, removes damaged unwanted mitochondria from cells through a lysosome-based degradation pathway to maintain cellular homeostasis. CSCs use mitophagy as a chief survival response mechanism for their growth, propagation, and tumorigenic ability. Mitochondrial biogenesis is a crucial cellular event replacing damaged mitochondria through the coordinated regulation of several transcription factors to achieve the bioenergetic demands of the cell. Because of the high mitochondrial content in CSCs, mitochondrial biogenesis is an interesting target to address the resistance mechanisms of anti-CSC therapy. However, to what extent both mitophagy and mitochondrial biogenesis are vital in promoting stemness, metabolic reprogramming, and drug resistance in CSCs has yet to be established. Therefore, in this review, we focus on understanding the interesting aspects of mitochondrial rewiring that involve mitophagy and mitochondrial biogenesis in CSCs. We also discuss their coordinated regulation in the elimination of CSCs, with respect to stemness and differentiation of the CSC phenotype, and the different aspects of tumorigenesis such as cancer initiation, progression, resistance, and tumor relapse. Finally, we address several other unanswered questions relating to targeted anti-CSC cancer therapy, which improves patient survival.

Secretory clusterin promotes oral cancer cell survival via inhibiting apoptosis by activation of autophagy in AMPK/mTOR/ULK1 dependent pathway.

AIMS: Secretory clusterin (sCLU) plays an important role in tumor development and cancer progression. However, the molecular mechanisms and physiological functions of sCLU in oral cancer is unclear. We examined the impact of sCLU-mediated autophagy in cell survival and apoptosis inhibition in oral cancer. MAIN METHODS: Immunohistochemical analysis was performed to analyze protein expression in patient samples. Autophagy and mitophagy was studied by immunofluorescence microscopy and Western blot. The gain and loss of function was studied by overexpression of plasmid and siRNA approaches respectively. Cellular protection against nutrient starvation and therapeutic stress by sCLU was studied by cell viability, caspase assay and meta-analysis. KEY FINDINGS: The data from oral cancer patients showed that the expression levels of sCLU, ATG14, ULK1, and PARKIN increased in grade-wise manners. Interestingly, sCLU overexpression promoted autophagy through AMPK/Akt/mTOR signaling pathway leading to cell survival and protection from long exposure serum starvation induced-apoptosis. Additionally, sCLU was demonstrated to interact with ULK1 and inhibition of ULK1 activity by SBI206965 was found to abolish sCLU-induced autophagy indicating critical role of ULK1 in induction of autophagy. Furthermore, sCLU was observed to promote expression of mitophagy-associated proteins in serum starvation conditions to protect cells from nutrient deprivation. The meta-analysis elucidated that high CLU expression is associated with therapy resistance in cancer and we demonstrated that sCLU-mediated mitophagy was revealed to inhibit cell death by cisplatin. SIGNIFICANCE: The present investigation has highlighted the probable implications of the clusterin-induced autophagy in cell survival and inhibition of apoptosis in oral cancer.

Bioactive compounds from marine invertebrates as potent anticancer drugs: the possible pharmacophores modulating cell death pathways.

Marine invertebrates are extremely diverse, largely productive, untapped oceanic resources with chemically unique bioactive lead compound contributing a wide range of screening for the discovery of anticancer compounds. The lead compounds have unfurled an extensive array of pharmacological properties owing to the presence of polyphenols, alkaloids, terpenoids and other secondary metabolites. The antioxidant, immunomodulatory and anti-tumor activities exhibited, are possibly regulated by the apoptosis induction, scavenging of ROS and modulation of cellular signaling pathways to defy the cellular deafness during carcinogenesis. Despite the enriched bioactive compounds, the marine invertebrates are largely unexplored as identification, screening, pre-clinical and clinical assessment of lead compounds and their synthetic analogs remain a major task to be solved. In the current review, we focus on the principle strategy and underlying mechanisms deployed by the bioactive anticancer compounds derived from marine invertebrates to combat cancer with special insight into the cell death mechanism.

Bacopa monnieri inhibits apoptosis and senescence through mitophagy in human astrocytes.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a potent neurotoxic agent that is responsible for impaired neuronal development and is associated with aging. Here, it was demonstrated that extracts of Bacopa monnieri (BM), a traditional Ayurvedic medicine, diminished the B[a]P-induced apoptosis and senescence in human astrocytes. BM was demonstrated to protect the immortalized primary fetal astrocytes (IMPHFA) from B[a]P-induced apoptosis and senescence by reducing the damaged mitochondria that produced reactive oxygen species (ROS). Furthermore, it was shown that B[a]P-triggered G2 arrest could be altered by BM, thus indicating that BM could reverse the cell cycle arrest and mediate a normal cell cycle in IMPHFA cells. In addition, the lifespan of Caenorhabditis elegans was assessed, which confirmed these effects in the presence of BM, compared to the B[a]P-treated group. Furthermore, the anti-senescence and anti-apoptotic activities of BM were observed to be mediated through the protective effect of mitophagy, and inhibition of mitophagy could not protect the astrocytes from mitochondrial ROS-induced apoptosis and senescence in BM-treated cells. Moreover, it was revealed that BM induced Parkin-dependent mitophagy to exert its cytoprotective activity in IMPHFA cells. In conclusion, the anti-senescence and anti-apoptotic effects of BM in astrocytes could combat pollution and aging-related neurological disorders.

Terminalia bellirica extract induces anticancer activity through modulation of apoptosis and autophagy in oral squamous cell carcinoma.

Terminalia bellirica (TB) has been used in traditional Indian medical system, Ayurveda. However, the mechanism underlying the efficacy of the TB extract against oral squamous cell carcinoma (OSCC) is yet to be explored. The present study established a connecting link between the TB extract induced apoptosis and autophagy in relation to reactive oxygen species (ROS). Our study revealed, that gallic acid in the TB extract possess a strong free radical scavenging capacity contributing towards the selective anti-proliferative activity. Furthermore, TB extract markedly enhanced the accumulation of ROS that facilitated mitochondrial apoptosis through DNA damage, indicating ROS as the vital component in regulation of apoptosis. This effect was effectively reversed by the use of a ROS scavenger, N-acetyl cysteine (NAC). Moreover, it was observed to induce autophagy; however, it attenuated the autophagosome-lysosome fusion in Cal33 cells without altering the lysosomal activity. Pharmacological inhibitors of autophagy, namely, 3-methyladenine and chloroquine, were demonstarated to regulate the stage-specific progression of autophagy post treatment with the TB extract, favouring subsequent activation of apoptosis. These findings revealed, presence of gallic acid in TB extract below NOAEL value causes oxidative upset in oral cancer cells and promote programmed cell death which has a potential therapeutic value against oral squamous cell carcinoma.