The Neem Limonoid Nimbolide Modulates Key Components of the DNA Damage Response Signalling in Cellular and Animal Models of Oral Squamous Cell Carcinoma

BACKGROUND: Deregulated DNA damage response (DDR) network is implicated in cancer progression and therapy resistance. OBJECTIVE: The present study was designed to investigate whether nimbolide, an anticancer neem limonoid, targets key components of the DDR signalling pathway in cellular and animal models of oral squamous cell carcinoma (OSCC). METHODS: OSCC cells (SCC-4 and SCC-9), 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinoma model, chemoresistant OSCC patient-derived xenograft (PDX) model established in athymic nude mice, and tissue sections from patients with oral premalignant/malignant disease were used for the study. Key molecules that orchestrate the DDR, including the MRN complex, ATM, DNA-PKcs, H2AX, and p53, were analysed by qRT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry. Cell proliferation and apoptosis indices were evaluated. RESULTS: Nimbolide significantly reduced 8-oxodG levels, expression of MRN, ATMS1891, and γ-H2AX, with an increase in p-p53S15 in OSCC cells as well as in the HBP model. Nimbolide potentiated the effect of KU-55933 in ATM inhibition. In the PDX model, nimbolide suppressed tumor formation, stimulated DDR and apoptosis, inhibited cell proliferation, and enhanced sensitivity to cisplatin. Analysis of p-ATM expression revealed a significant increase during the sequential progression of hamster and human OSCC. CONCLUSIONS: This study provides compelling evidence that nimbolide functions as a DDR inhibitor in cellular and hamster OSCC models and as a DDR activator in the PDX model primarily by targeting ATM. Small molecules like nimbolide that modulate DDR are of immense benefit in cancer therapy. The study has also unveiled p-ATM as a promising biomarker of tumour progression in human OSCCs.

Limonoids from neem (Azadirachta indica A. Juss.) are potential anticancer drug candidates.

Neem (Azadirachta indica A. Juss.), a versatile evergreen tree recognized for its ethnopharmacological value, is a rich source of limonoids of the triterpenoid class, endowed with potent medicinal properties. Extracts of neem have been documented to display anticancer effects in diverse malignant cell lines as well as in preclinical animal models that has largely been attributed to the constituent limonoids. Of late, neem limonoids have become the cynosure of research attention as potential candidate agents for cancer prevention and therapy. Among the various limonoids found in neem, azadirachtin, epoxyazadiradione, gedunin, and nimbolide, have been extensively investigated for anticancer activity. Azadirachtin, a potent biodegradable pesticide, exhibits profound antiproliferative effects by preventing mitotic spindle formation and cell division. The antiproliferative activity of gedunin has been demonstrated to be mediated primarily via inhibition of heat shock protein90 and its client proteins. Epoxyazadiradione inhibits pro-inflammatory and kinase-driven signaling pathways to block tumorigenesis. Nimbolide, the most potent cytotoxic neem limonoid, inhibits the growth of cancer cells by regulating the phosphorylation of keystone kinases that drive oncogenic signaling besides modulating the epigenome. There is overwhelming evidence to indicate that neem limonoids exert anticancer effects by preventing the acquisition of hallmark traits of cancer, such as cell proliferation, apoptosis evasion, inflammation, invasion, angiogenesis, and drug resistance. Neem limonoids are value additions to the armamentarium of natural compounds that target aberrant oncogenic signaling to inhibit cancer development and progression.

Crosstalk between PD-L1 and Jak2-Stat3/ MAPK-AP1 signaling promotes oral cancer progression, invasion and therapy resistance.

BACKGROUND: Programmed cell death ligand-1 (PD-L1)is an antitumor immunity molecule and a great target to cure oral cancer; nonetheless, the limited success can be attributed to many complex pathways and tumor-related interferences. METHODS: In the present study, 150 human oral squamous cell carcinoma (OSCC) tissue samples, including 17 adjacent normals, 56 primary tumors, 47 invasive tumors, and 30 therapy-resistant (RT) samples, were included. The parental/cisplatin-resistant (CisR-SCC4/9) cells were utilized for overexpression (Jak1-3 wild type and catalytically inactive), knockdown (PD-L1 siRNA), targeting MAPK/PI3K/Jak-Stat pathways (SMIs) and checking microsomes. The expression of PD-L1, transcription factors (TFs), signaling pathways, survival/apoptosis, therapy resistance, and invasiveness-related molecules/their activity were determined by RT-PCR, Immunohistochemistry, Western blot, Gelatin Zymography, and MTT assay. RESULTS: Advanced OSCC tumors (invasive and drug-resistance), CisR-SCC4/9 cells, and secretory exosomes (CisR-SCC4/9) were found with increased PD-L1 expression. PD-L1 mRNA/protein showed a positive correlation with different TFs (AP1 > Stat3 > c-myc > NFκB) in tumor samples. The PD-L1 expression was more influenced by Jak-Stat/ MAPK-AP1 pathways over PI3K. The ectopic expression of Jak1-3 suggests Jak2 inducted PD-L1 level over Jak1/Jak3. Finally, PD-L1 directly supports survival (Bcl-xL, Bax, cleaved caspase-3), invasion (MMP2/9), and drug-resistance (ALDH-1A1/-3A1) program in OSCC through its link with several molecules. CONCLUSIONS: PD-L1 was regulated mainly by the Jak2-Stat3/ MAPK-AP1 pathway, and besides the routine immunological functions, it supports OSCC survival, invasion, and therapy resistance. PD-L1 can be used as an indicator of severity and can be targeted along with Jak2-Stat3/ MAPK-AP1 for a better outcome OSCC.

Obesity Associated with Prediabetes Increases the Risk of Breast Cancer Development and Progression-A Study on an Obese Rat Model with Impaired Glucose Tolerance.

Patients with comorbidities of obesity and diabetes are recognized to be at high risk of breast cancer development and face worse breast cancer outcomes. Though several reports showed the reinforced link between obesity, diabetes, and prediabetes with breast cancer, the underlying molecular mechanisms are still unknown. The present study aimed to investigate the underlying molecular link between increased risks of breast cancer due to coincident diabetes or obesity using a spontaneous obese rat model with impaired glucose tolerance (WNIN/GR-Ob rat). A single dose of solubilized DMBA suspension (40 mg/kg body weight) was orally administered to the animals at the age of 60 days to induce breast tumors. The tumor incidence, latency period, tumor frequency, and tumor volume were measured. Histology, immunohistochemistry, and immunoblotting were performed to evaluate the tumor morphology and expression levels of signal molecules. The development of mammary tumors in GR-Ob rats was characterized by early onset and shorter latency periods compared to control lean rats. While 62% of obese rats developed breast tumors, tumor development in lean rats was only 21%. Overexpression of ER, PR, Ki67, and p53 markers was observed in tumor tissues of obese rats in comparison with lean rats. The levels of the hallmarks of cell proliferation and angiogenesis involved in IGF-1/PI3K/Akt/GSK3ß/ß-catenin signaling pathway molecules were upregulated in obese rat breast tumors compared to lean rats. Furthermore, obesity with prediabetes is associated with changes in IGF-1 signaling and acts on PI3K/Akt/GSK3ß/ß-catenin signaling, which results in rapid cell proliferation and development of breast tumors in obese rats than the lean rats. These results indicate that tumor onset and development were faster in spontaneous obese rat models with impaired glucose tolerance than in their lean counterparts.

Utility of Prostate-Specific Antigen Isoforms and Prostate Health Index in the Diagnosis of Metastatic Prostate Cancer.

Objective The current study was undertaken to investigate the utility of total prostate-specific antigen (tPSA), its isoform [-2] proPSA (p2PSA), and prostate health index (PHI) in the diagnosis of metastatic prostate cancer (PCa). Materials and Methods This study was conducted from March 2016 to May 2019. Eighty-five subjects who were diagnosed with PCa for the first time, following transrectal ultrasound-guided prostate biopsy, were included in the study. The prebiopsy blood samples were analyzed in Beckman Coulter Access-2 Immunoanalyzer for tPSA, p2PSA, and free PSA (fPSA), and the calculated parameters included %p2PSA, %fPSA, and PHI. Mann-Whitney's U test was used as test of significance, and p -value less than 0.05 was considered statistically significant. Results Of the 85 participants, 81.2% ( n = 69) had evidence of metastasis, both clinically and pathologically. The median tPSA (ng/mL), p2PSA (pg/mL), %p2PSA, and PHI were significantly higher in the group with evidence of metastasis (46.5 vs. 13.76; 198.0 vs. 35.72; 3.25 vs. 1.51; 237.58 vs. 59.74, respectively). The sensitivity (%), specificity (%), negative predictive value (%), and positive predictive value (%) to diagnose metastatic PCa of tPSA at a cutoff of 20 ng/mL, PHI at a cutoff of 55, and %p2PSA at a cutoff of 1.66 were 92.7, 98.5, and 94.2; 37.5, 43.7, and 62.5; 54.5, 87.5, and 71.4; and 86.4, 88.3, and 91.5, respectively. Conclusion Using tests such as %p2PSA and PHI in the standard armamentarium for the diagnosis of metastatic PCa in addition to PSA will help in selecting the appropriate treatment strategy, including active surveillance.

Role and regulation of GLUT1/3 during oral cancer progression and therapy resistance.

OBJECTIVE: This study aimed to determine whether glucose transporter-1/3 (GLUT1/3) increased expression could contribute to oral tumor severity. Furthermore, this study detected whether GLUT1/3 mRNA/protein was associated with oncogenic transcription factors (HIF1α, AP1 and NFκB) and whether by blocking GLUT1 along with cisplatin could sensitize drug-resistant OSCC cells. DESIGN: We used 120 post-operated human tissue samples, including 35 primary tumors (PT), 43 invasive tumors (N1-3), 17 recurrent chemoradiation-resistant tumors (RCRT), and 25 PT-adjacent normal tissues (AN). The cisplatin-resistant (CisR-SCC4/9) cells were generated using a drug escalation strategy from parental SCC4/9 cells. The BAY-876 treatment blocked GLUT1 in OSCC cells. Western Blot, Immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect various proteins and mRNA. Cell survival was determined by MTT assay. RESULTS: GLUT1/3 expression was observed more in PT over AN tissue (PT > AN), N1-3 > PT, and .RCRT > PT. GLUT1 expression was maximum in the RCRT group and CisR-SCC4/9 cells over their parental counterpart, linked with tumor size (p=0.0037) and loco-regional invasiveness (p=0.0422). GLUT1/3 mRNA/protein was correlated (positively) with oncogenic transcription factors (TFs) like HIF1α, AP1 and NFκB. We found the degree of positive correlation of these TFs with GLUT1/3 was in the order c-Jun > HIF1α > Fra-2 > NFκB > c-Fos. Treatment of BAY-876 and cisplatin-induced cell death in both CisR-SCC4/9 cells, possibly by triggering apoptosis and autophagy. CONCLUSION: Collectively, our results demonstrated increased GLUT1/3 overexpression linked with oral tumor severity like invasion and therapy resistance, and it was powered mainly by c-Jun (AP1). Blocking GLUT1 receptors and cisplatin application can sensitize CisR-OSCC cells.

Nimbolide, a Neem Limonoid, Inhibits Angiogenesis in Breast Cancer by Abrogating Aldose Reductase Mediated IGF-1/PI3K/Akt Signalling.

BACKGROUND & OBJECTIVE: The insulin/IGF-1R/PI3K/Akt signalling cascade is increasingly being linked to breast cancer development, with aldose reductase (AR) playing a key role in mediating the crosstalk between this pathway and angiogenesis. The current study was designed to investigate whether nimbolide, a neem limonoid, targets the oncogenic signaling network to prevent angiogenesis in breast cancer. METHODS: Breast cancer cells (MCF-7, MDA-MB-231), EAhy926 endothelial cells, MDA-MB-231 xenografted nude mice, and tumour tissues from breast cancer patients were used for the study. The expression of AR and key players in IGF-1/PI3K/Akt signaling and angiogenesis was evaluated by qRT-PCR, immunoblotting, and immunohistochemistry. Molecular docking and simulation, overexpression, and knockdown experiments were performed to determine whether nimbolide targets AR and IGF-1R. RESULTS: Nimbolide inhibited AR with consequent blockade of the IGF-1/PI3K/Akt and /HIF-1alpha/VEGF signalling circuit by influencing the phosphorylation and intracellular localisation of key signaling molecules. The downregulation of DNMT-1, HDAC-6, miR-21, HOTAIR, and H19 with the upregulation of miR-148a/miR-152 indicated that nimbolide regulates AR and IGF-1/PI3K/Akt signaling via epigenetic modifications. Coadministration of nimbolide with metformin and the chemotherapeutic drugs tamoxifen/cisplatin displayed higher efficacy than single agents in inhibiting IGF-1/PI3K/Akt/AR signaling. Grade-wise increases in IGF-1R and AR expression in breast cancer tissues underscore their value as biomarkers of progression. CONCLUSION: This study provides evidence for the anticancer effects of nimbolide in cellular and mouse models of breast cancer besides providing leads for new drug combinations. It has also opened up avenues for investigating potential molecules such as AR for therapeutic targeting of cancer.

Glycogen synthase kinase-3ß inactivation promotes cervical cancer progression, invasion, and drug resistance.

Human papillomavirus (HPV) infection-dependent cervical cancer is one of the most common gynecological cancers and often becomes aggressive, with rapid proliferation, invasion/migration, and drug resistance. Here, 135 fresh human cervical squamous cell carcinoma (CSCC) tissue specimens, comprising 21 adjacent normal (AN), 30 cervical intraepithelial neoplasia (CIN1-3 ), 45 CSCC, and 39 drugs (chemo-radiation)-resistant cervical tumor (DRCT) tissues were included. HPV-positive (HeLa, SiHa), HPV-negative (C33A), and cisplatin-resistant (CisR-HeLa/-SiHa/-C33A) cell lines were used for in vitro studies. HPV16/18 oncoproteins E6/E7, pERK1/2, and glycogen synthase kinase-3 (GSK3) and the matrix metalloproteinases (MMPs) MMP-9/-2 were assessed using immunohistochemistry, WB, and gelatin zymography. HPV16/18 infection was observed in 16.7% of the CIN1-3 , 77.8% of the CSCC, and 89.7% of DRCT samples. Total and inactive GSK3ß expressions were associated with overall CSCC progression (p = 0.039 and p = 0.024, respectively) and chemoresistance (p = 0.004 and p = 0.014, respectively). Positive correlations were observed, between the expression of E6 and pGSK3ß expression (p = 0.013); E6 and CSCC progression (p < 0.0001)/drug resistance (p = 0.0001). CisR-HeLa/-SiHa was more dependent on pGSK3ß, and activation of GSK3 by SMIs (iAkt), treatment with nimbolide, or knockdown of E6/E7 reduced cisplatin resistance and promoted apoptosis. Hence, the activation of GSK3ß with nimbolide and iAkt can be exploited for therapeutic interventions of cervical cancer.

Nimbolide, a Neem Limonoid, Is a Promising Candidate for the Anticancer Drug Arsenal.

Nimbolide, a major limonoid constituent of Azadirachta indica, commonly known as neem, has attracted increasing research attention owing to its wide spectrum of pharmacological properties, predominantly anticancer activity. Nimbolide is reported to exert potent antiproliferative effects on a myriad cancer cell lines and chemotherapeutic efficacy in preclinical animal tumor models. The potentiality of nimbolide to circumvent multidrug resistance and aid in targeted protein degradation broaden its utility in enhancing therapeutic modalities and outcome. Accumulating evidence indicates that nimbolide prevents the acquisition of cancer hallmarks such as sustained proliferation, apoptosis evasion, invasion, angiogenesis, metastasis, and inflammation by modulating kinase-driven oncogenic signaling networks. Nimbolide has been demonstrated to abrogate aberrant activation of cellular signaling by influencing the subcellular localization of transcription factors and phosphorylation of kinases in addition to influencing the epigenome. Nimbolide, with its ever-expanding repertoire of molecular targets, is a valuable addition to the anticancer drug arsenal.

Pluripotency transcription factor Nanog and its association with overall oral squamous cell carcinoma progression, cisplatin-resistance, invasion and stemness acquisition.

BACKGROUND: Cisplatin-resistant oral squamous cell carcinoma (OSCC) cells acquire stem-like characteristics and are difficult to treat. Nanog is a transcription factor and needed for maintenance of pluripotency, but its transcription-promoting role in OSCC progression and cisplatin resistance is poorly understood. METHODS: Here, 110 fresh human tissue specimens of various stages, including invasive (N1-3 )/chemoradiation-resistant OSCC samples, cisplatin-resistant (CisR-SCC-4/-9) OSCC cells/parental cells, photochemical ECGC, and siRNA (Nanog) were used. RESULTS: Nanog overexpression was associated with overall progression, chemoresistance, and invasion of OSCC. Nanog recruitment to c-Myc, Slug, E-cadherin, and Oct-4 gene promoter was observed. Positive correlation of Nanog protein expression with c-Myc, Slug, cyclin D1, MMP-2/-9, and Oct-4 and negative correlation with E-cadherin gene expression were found. Knockdown of Nanog and treatment of epicatechin-3-gallate reversed cisplatin resistance and diminished invasion/migration potential. CONCLUSION: Nanog directly participated in the regulation of Slug, E-cadherin, Oct-4, and c-Myc genes, causing cisplatin resistance/recurrence of OSCC.

Activation of PI3K/Akt/NF-kB Signaling Mediates Swedish Snus Induced Proliferation and Apoptosis Evasion in the Rat Forestomach: Modulation by Blueberry.

BACKGROUND AND OBJECTIVES: The present study was undertaken to ascertain whether the modulatory effects of blueberries on cell proliferation induced by Swedish snus in the rat forestomach epithelium is mediated via abrogation of the PI3K/Akt/NFκB signaling axis that regulates cell fate decision. METHODS: The transcript and protein expression of genes involved in cell cycle progression and apoptosis, as well as canonical PI3K/Akt/NF-κB signaling pathways, were analyzed by qRT-PCR, immunoblotting and ELISA. Expression profiling of noncoding RNAs (ncRNAs) that influence PI3K/Akt/NF-κB signaling was undertaken. TUNEL assay was performed using flow cytometry. RESULTS: Administration of snus induced basal cell hyperplasia in the rat forestomach with increased cell proliferation and inhibition of apoptosis. This was associated with the activation of PI3K/Akt/NFκB signaling. Coadministration of blueberries significantly suppressed snus-induced hyperplasia. Analysis of the molecular mechanisms revealed that blueberries suppress the phosphorylation of Akt, NF-κB and IKKß, prevent nuclear translocation of NF-κB and modulate the expression of microRNAs that influence PI3K/Akt/NF-κB signaling. CONCLUSION: Taken together, the results of the current study provide compelling evidence that blueberries exert significant protective effects against snus-induced soft tissue changes in the rat forestomach epithelium mediated by inhibiting key molecular players in the PI3K/Akt/NF-κB signaling axis. Long-term studies on the impact of snus exposure on various cellular processes, signaling pathways, and the interplay between genetic and epigenetic mechanisms are however warranted. The results of this investigation may contribute to the development of protection against soft tissue changes induced by smokeless tobacco in the human oral cavity.

Abrus agglutinin inhibits oral carcinogenesis through inactivation of NRF2 signaling pathway.

Abrus agglutinin (AGG), a heterotetrameric type II ribosome inactivating protein isolated from the seeds of Abrus precatorius shows potent antitumor activity in different cancer models. We examined the role of antioxidant system in modulation of the anticancer activity of AGG in in vitro and in hamster model of oral cancer. AGG promotes apoptosis through accumulation of ROS in CAL33 cells. Interestingly, our data showed that AGG decreases the activity of antioxidant enzymes including superoxide dismutase, catalase, glutathione peroxidase in CAL33 cells indicating antioxidant enzyme inhibition leads to AGG-induced ROS accumulation. Moreover, AGG inhibits expression of NRF2, transcription factor which regulates the expression of antioxidant enzymes in CAL33 cells. We found that AGG induces autophagy stimulation and loss of p62 expression in CAL33 cells. Furthermore, it showed that NRF2 expression is restored in the presence of 3-methyladenine and Baficomycin-A1 establishing role of autophagy in modulation of NRF2 through p62. Our study showed that AGG significantly inhibited tumor growth in DMBA-induced carcinogenesis. In immunohistochemical analysis, AGG-treated tumor displays higher caspase 3 expression and less p62 and NRF2 expression in comparison to the control. In conclusion, AGG-induced degradation of NRF2 through autophagy leads to ROS accumulation dependent apoptosis which might be used for treatment of oral cancer.

Astaxanthin inhibits hallmarks of cancer by targeting the PI3K/NF-κΒ/STAT3 signalling axis in oral squamous cell carcinoma models.

Aberrant activation of the PI3K/Akt signalling pathway, a major driving force of diverse cellular processes has been implicated in tumour development and progression. Here, we report that astaxanthin (AXT), a potent antioxidant ketocarotenoid prevents cancer hallmarks by inhibiting PI3K/Akt and the associated downstream NF-κB and STAT-3 signalling pathways in SCC131 and SCC4 oral cancer cells as well as in the hamster buccal pouch carcinogenesis model. Using small molecule inhibitors of NF-κB, STAT-3 and PI3K and by overexpression of PI3K, we provide evidence to show that AXT inhibits NF-κB and STAT-3 signalling and cancer hallmarks by restraining the kinase activity of PI3K/Akt. Additionally, AXT downregulated the noncoding RNAs (ncRNAs), miR-21 and HOTAIR that influence PI3K/Akt signalling emphasising its modulatory effects on epigenetic regulation. Ethyl cellulose-based AXT nanoparticles showed greater chemotherapeutic efficacy in the hamster oral carcinogenesis model compared to native AXT. We suggest that AXT prevents cell proliferation, apoptosis evasion, invasion and angiogenesis by intercepting the crosstalk between the PI3K/Akt, NF-κB and STAT-3 signalling circuits both in vitro and in vivo. Astaxanthin that abrogates the PI3K/Akt signalling axis, a central hub that orchestrates acquisition of cancer hallmarks is a promising candidate for anticancer drug development.

Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase is an archetypal multifunctional moonlighting protein involved in diverse cellular processes including metabolism, insulin signaling, proliferation, differentiation, apoptosis, neuronal function and embryonic development. The two known isoforms, GSK-3α and GSK-3ß that undergo activation/inactivation by post-translational, site-specific phosphorylation incorporate a vast number of substrates in their repertoire. Dysregulation of GSK-3 has been linked to diverse disease entities including cancer. The role of GSK-3 in cancer is paradoxical and enigmatic. The enzyme functions as a tumour promoter or suppressor based on the context, cell type and phosphorylation status. GSK-3 is the central hub that orchestrates signals from the Wnt/ß-catenin, PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, hedgehog, Notch and TP53 pathways to elicit regulatory influences on cancer initiation, epithelial-mesenchymal transition, and resistance to therapy. As a direct target of several microRNAs, GSK-3 influences hallmark attributes of cancer, cancer stemness and treatment resistance. There is overwhelming evidence to indicate that GSK-3 is aberrantly regulated in different cancer types. Consequently, GSK-3 has emerged as a potential therapeutic target in cancer. A plethora of natural and synthetic GSK-3 modulators have been discovered and the number of patents published for GSK-3 inhibitors has also been steadily increasing in recent years. This review focuses on the intricate interactions between GSK-3 and oncogenic signalling circuits as well as the feasibility of targeting GSK-3 for the treatment of cancer.

Role and regulation of proapoptotic Bax in oral squamous cell carcinoma and drug resistance.

BACKGROUND: Bax, a proapoptotic protein but its regulation during oral cancer progression and resistance remains elusive. METHODS: A total of 127 samples including adjacent normal, primary tumor, and resistance to chemoradiation therapy (RCRT) samples from oral squamous cell carcinoma (OSCC) patients were used. The status of Bax was analyzed at DNA/mRNA/protein levels and the results were correlated with p53 and Akt expression in tissue samples/cisplatin-resistant oral tongue SCC (SCC9/SCC4-CisR) cell line. RESULTS: Frequent progressive decrease of Bax expression with infrequent promoter methylation, polymorphisms G(-248)A, and mutations was observed in OSCC progression/resistance. Furthermore, by targeting Akt pathway, induction of Bax-dependent cell death was observed and this was further enhanced with nimbolide treatment in SCC9/SCC4-CisR cells. CONCLUSION: Hence, the Bax gene alteration and its deregulation through p53/Akt pathway are important for OSCC progression and drug resistance. Akt Inhibitor VIII and nimbolide synergistically induce Bax, and it is therefore beneficial for chemosensitizing cisplatin-resistant human OSCC.

Impact of stainless-steel welding fumes on proteins and non-coding RNAs regulating DNA damage response in the respiratory tract of Sprague-Dawley rats.

Substantial evidence has established the negative impact of inhalation exposure to welding fumes on respiratory functions. The aim of the present study was to investigate the effect of welding fume inhalation on expression of molecules that function as sensors, transducers and effectors of DNA damage response (DDR) in the respiratory tract of male Sprague-Dawley rats. Animals were exposed to 50 mg/m3 stainless steel welding fumes for 1 h/d for 4, 8, and 12 weeks, respectively. Histological examination demonstrated preneoplastic changes in trachea and bronchi with focal atelectasis and accumulation of chromium (Cr) in the lungs. This was associated with elevated levels of DNA damage markers (8-oxodG, γH2AX), ATM phosphorylation, cell cycle arrest, apoptosis induction, activation of homologous recombination (HR), non-homologous end joining (NHEJ), and Nrf2 signaling, as well as altered expression of noncoding RNAs (ncRNAs). However, after 12 weeks of exposure, DDR was compromised as reflected by resumption of the cell cycle, repair inhibition, and failure of apoptosis. Data demonstrate that exposure to welding fumes influences two crucial layers of DDR regulation, phosphorylation of key proteins in NHEJ and HR, as well as the ncRNAs that epigenetically modulate DDR. Evidence indicates that marked DNA damage coupled with non-productive DNA repair and apoptosis avoidance may be involved in neoplastic transformation.

Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3ß signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer.

BACKGROUND: The cell-surface glycoprotein CD44 is an important oral cancer stem cell (OCSC) marker and plays significant role in oral squamous cell carcinoma (OSCC) aggressiveness, however, the regulation of CD44 is incompletely understood. METHODS: In the present study, 145 fresh human OSCC tissue specimens, including 18 adjacent normal, 42 noninvasive (N0), 53 invasive tumor samples (N1-3) and 32 chemo-radiation resistant samples (RCRT), were included. The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of pERK1/2, GSK3ß, NICD (Notch) pathways; the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) cells, were used. Knock down of CD44v4/CD44v6 (by siRNA) or inactivation of MAPK/PI3K pathways using specific PD98059/LY294002 was achieved for in vitro analysis of chemoresistance and invasion/migration. RESULTS: Elevated CD44 variants were associated with overall OSCC progression, chemoresistance and invasion. Positive correlations were observed, mainly between the expression of CD44v4 and the activation of ERK1/2 causing chemoresistance, whereas CD44v6 expression and inactivation of GSK3ß caused invasiveness of OSCC. Cisplatin resistant, CisR-SCC9/SCC4 cell lines showed OCSC properties. Inhibition of MEK/ERK1/2 by SMI or knock down (KD) of CD44v4 by siRNA reversed cisplatin-resistance, whereas blocking the PI3K/Akt/GSK3ß pathway by SMI or KD of CD44v6 isoforms by respective siRNA diminished invasion/metastasis potential. CONCLUSION: Collectively, our results demonstrated that CD44v4 expression is more linked with ERK1/2 activation and promote cisplatin resistance, whereas CD44v6 expression is associated primarily with PI3K/Akt/GSK3ß activation and driving tumor invasion/migration.

Nimbolide, a neem limonoid inhibits cytoprotective autophagy to activate apoptosis via modulation of the PI3K/Akt/GSK-3ß signalling pathway in oral cancer.

Of late, nimbolide, a limonoid from the neem tree (Azadirachta indica) has gained increasing research attention owing to its potent antiproliferative and apoptosis-inducing effects. The present study was designed to investigate the effect of nimbolide on autophagy and the time point at which the phosphorylation status of GSK-3ß and PI3K dictate the choice between autophagy and apoptosis in SCC131 and SCC4 oral cancer cells. Additionally, we analysed changes in the expression of proteins involved in autophagy and apoptosis after therapeutic intervention with nimbolide in a hamster model of oral oncogenesis. Furthermore, we also demonstrate changes in the expression of key genes involved in apoptosis and autophagy during the stepwise evolution of hamster and human OSCCs. Nimbolide-induced stereotypical changes in oral cancer cells characteristic of both apoptosis and autophagy. Time-course experiments revealed that nimbolide induces autophagy as an early event and then switches over to apoptosis. Nimbolide negatively regulates PI3K/Akt signalling with consequent increase in p-GSK-3ßTyr216, the active form of GSK-3ß that inhibits autophagy. Downregulation of HOTAIR, a competing endogenous RNA that sponges miR-126 may be a major contributor to the inactivation of PI3K/Akt/GSK3 signalling by nimbolide. Analysis of key markers of apoptosis and autophagy as well as p-AktSer473 during sequential progression of hamster and human OSCC revealed a gradual evolution to a pro-autophagic and antiapoptotic phenotype that could confer a survival advantage to tumors. In summary, the results of the present study provide insights into the molecular mechanisms by which nimbolide augments apoptosis by overcoming the shielding effects of cytoprotective autophagy through modulation of the phosphorylation status of Akt and GSK-3ß as well as the ncRNAs miR-126 and HOTAIR. Development of phytochemicals such as nimbolide that target the complex interaction between proteins and ncRNAs that regulate the autophagy/apoptosis flux is of paramount importance in cancer prevention and therapeutics.

Gedunin, A Neem Limonoid in Combination with Epalrestat Inhibits Cancer Hallmarks by Attenuating Aldose Reductase-Driven Oncogenic Signaling in SCC131 Oral Cancer Cells.

BACKGROUND AND OBJECTIVES: Aldose Reductase (AR), a polyol pathway enzyme that mediates diabetic complications is implicated in tumour development and progression. This study was undertaken to determine whether gedunin, a neem limonoid prevents the hallmarks of cancer by inhibiting AR and the associated downstream PI3K/Akt/mTOR/ERK/NF-κB signalling axis in the SCC131 oral cancer cell line. METHODS: The expression of AR and key molecules involved in cell proliferation, apoptosis, autophagy, invasion and angiogenesis was analysed by qRT-PCR, and immunoblotting. ROS generation and cell cycle were analysed by FACS. Alamar blue assay and scratch assay were used to evaluate cell proliferation and migration in the endothelial cell line Eahy926. RESULTS: Gedunin and the AR inhibitor epalrestat inhibited AR expression and ROS generation. Cell cycle arrest at G1/S was associated with cell death by autophagy with subsequent switch over to apoptosis. Furthermore, hypoxia-induced cell migration was inhibited in Eahy926 cells with downregulation of pro-invasive and proangiogenic proteins in SCC131 as well as Eahy926 cells. Co-inactivation of Akt and ERK was coupled with abrogation of IKK/NF-κB signaling. However, the combination of gedunin and epalrestat was more effective than single agents. CONCLUSION: Inhibition of AR-mediated ROS signalling may be a key mechanism by which gedunin and epalrestat exert their anticancer effects. Our results provide compelling evidence that the combination of gedunin and epalrestat modulates expression of key oncogenic signalling kinases and transcription factors primarily by influencing phosphorylation and subcellular localisation. AR inhibitors such as gedunin and epalrestat are novel candidate agents for cancer prevention and therapy.