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.

Oral tumor heterogeneity, its implications for patient monitoring and designing anti-cancer strategies.

Oral cancer tumors occur in the mouth and are mainly derived from oral mucosa linings. It is one of the most common and fatal malignant diseases worldwide. The intratumor heterogeneity (ITH) of oral cancerous tumor is vast, so it is challenging to study and interpret. Due to environmental selection pressures, ITH arises through diverse genetic, epigenetic, and metabolic alterations. The ITH also talks about peri-tumoral vascular/ lymphatic growth, perineural permeation, tumor necrosis, invasion, and clonal expansion/ the coexistence of multiple subclones in a single tumor. The heterogeneity offers tumors the adaptability to survive, induce growth/ metastasis, and, most importantly, escape antitumor therapy. Unfortunately, the ITH is prioritized less in determining disease pathology than the traditional TNM classifications or tumor grade. Understanding ITH is challenging, but with the advancement of technology, this ITH can be decoded. Tumor genomics, proteomics, metabolomics, and other modern analyses can provide vast information. This information in clinics can assist in understanding a tumor's severity and be used for diagnostic, prognostic, and therapeutic decision-making. Lastly, the oral tumor ITH can lead to individualized, targeted therapy strategies fighting against OC.

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.

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.

Polymorphisms and haplotypes of TLR-4/9 associated with bacterial infection, gingival inflammation/recession and oral cancer.

BACKGROUND: The expression and SNPs of innate immunity genes TLR-4/9 for bacterial infection, gingival inflammation/gingival recession (GIGR), and oral squamous cell carcinoma (OSCC) are largely unknown. PATIENTS AND METHOD: 235 specimens (120 OSCC cases, among which 85 cases with either Porphyromonas gingivalis, Fusobacterium nucleatum or Treponema denticola infection and GIGR) and 115 healthy controls were used to know the expression and polymorphisms (TLR-4: N1:rs10759931, N2:rs11536889, N3:rs1927911, N4:rs4986790; TLR-9: N5:rs5743836, N6:rs352140, N7:rs187084 and N8:rs352139) of TLR-4/9 by western blot, RT-PCR, and allele-specific (AS)-PCR followed by sequencing. RESULTS: Increased TLR-4/9 mRNA/protein expression, bacterial infection (BI) and GIGR were associated with OSCC incidence. One of the three BI and GIGR was observed in 70.83% of OSCC cases, whereas all the HC used were free from any of these three BI/GIGR. The N3: CT-genotype (Odds Ratio hereafter as O.R.=1.811, p = 0.0338), TT-genotype (O.R.=3.094, p = 0.0124), 'T'-allele (O.R.=1.821, p = 0.003), N4: AG-genotype (O.R.=2.015, p = 0.0222) and 'G'-allele (O.R.=1.86, p = 0.018) of TLR-4 as well as the N5: CC-genotype (O.R.=3.939, p = 0.0017), 'C'-allele (O.R.=1.839, p = 0.0042), N6: AA-genotype (O.R.=2.195, p = 0.0234), 'A'-allele (O.R.=1.569, p = 0.0163), N7: TC-genotype (O.R.=2.083, p = 0.0136), CC-genotype (O.R.=2.984, p = 0.003) and 'C'-allele (O.R.=1.885, p = 0.0008) of TLR-9 were associated with increased OSCC risk. Similarly, the N2:'C'-allele (O.R.=1.615, p = 0.0382), N3: TT-genotype (O.R.=2.829, p = 0.0336), 'T'-allele (O.R.=1.742, p = 0.0115), N4: AG-genotype (O.R.=2.221, p = 0.0147) and 'G'-allele (O.R.=1.890, p = 0.0238) of TLR-4 as well as the N5: CC-genotype (O.R.=2.830, p = 0.031), N6: AA-genotype (O.R.=2.6, p = 0.0122) and 'A'-allele (O.R.=1.746, p = 0.0064), N7:CC-genotype (O.R.2.706, p = 0.0111) and 'C'-allele (O.R. 1.774, p = 0.0055) of TLR-9 were correlated with GIGR and BI. TLR-4 (N1-N2-N3-N4: A-C-T-A (O.R.=2.1, p = 0.0069) and TLR-9 (N5-N6-N7-N8: T-A-C-A (O.R.=2.019, p = 0.0263); C-A-C-A (O.R.=6.0, p = 0.0084); C-A-C-G (O.R.=4.957, p = 0.0452) haplotypes were linked with OSCC vulnerability, while the TLR-4 (N1-N2-N3-N4: G-C-C-A (O.R.=0.5752, p = 0.0131) and TLR-9 (N5-N6-N7-N8: T-G-T-A (O.R.=0.5438, p = 0.0314); T-G-T-G (O.R.=0.5241, p = 0.036) haplotypes offered protection. CONCLUSION: TLR-4/9 expression, polymorphisms, and BI-induced GIGR could increase OSCC risk. This may be used in pathogenesis and oral cancer prediction.

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.

ERK-mediated upregulation of matrix metalloproteinase-2 promotes the invasiveness in human oral squamous cell carcinoma (OSCC).

BACKGROUND: Loco-regional invasion is commonly found in oral squamous cell carcinoma (OSCC) and is associated with its poor survival rate. Matrix metalloproteinase-2 (MMP-2) has been implicated in OSCC progression, but its regulation is poorly understood. MATERIALS AND METHODS: Here, one hundred twenty-seven different post-operated human oral cancer tissue samples were analyzed. The messenger RNA (mRNA) expression, protein expression, and MMP-2 activity and MT1-MMP, TIMP-2, and TFs (NFκB, AP1, Sp1, and Twist) were observed semi-quantitative RT-PCR, western blotting, and gelatin zymography. In addition, OSCC derived Cal-27, SCC4/9 cells, photochemical ECGC, and MAPK-pathway inhibitor PD98059 were utilized for in vitro testing and wound healing assay. RESULT: s: Increased protein and activity level of MMP-2 was detected in non-invasive (N0) and invasive (N1-3) oral tumors as compared to the control (adjacent normal) samples. MMP-2 protein and mRNA expression were positively associated with the TFs and MT1-MMP, negatively associated with TIMP-2 expression. Similarly, the MMP-2 expression/activity was related to several signal-transduction pathways like ERK1/2 and wnt-ß-catenin pathways. Treatment of ECGC/MEK inhibitor (PD98059) diminished MMP-2 activity and invasion/migration potential in OSCC. CONCLUSION: Our research suggests that the ERK1/2 driven overexpression/activation of MMP-2 was linked with the overall OSCC invasion and metastasis. Treatment of MEK inhibitor (PD98059) and ECGC diminished MMP-2 activity and thus could be exploited as a therapeutic strategy to control the invasive OSCC.

Bcl-xL expression and regulation in the progression, recurrence, and cisplatin resistance of oral cancer.

BACKGROUND: Bcl-xL is an anti-apoptotic molecule, but its role in the progression and recurrent/ drug-resistant oral squamous cell carcinoma (OSCC) is poorly understood. MATERIALS AND METHODS: A total of one hundred twenty-five human OSCC tissue specimens including twenty-nine adjacent normals (AN), sixty-nine primary tumors (PT), twenty-seven recurrent chemoradiation resistance (RCRT) samples, and oral tongue SCC derived cisplatin-resistant (CisR SCC-4/-9) cells were used, for this study. Protein/mRNA expression levels of Bcl-xL and its regulation by ERK1/2, Stat-3, p53, NFκB, AP-1 (components: c-Jun, c-Fos, and Fra-2) molecules, and cell viability were measured by immunohistochemistry, Western blot, RT-PCR, and MTT analysis. Further, the individual and synergistic effects of Fra-2 (siRNA) and nimbolide were tested in CisR SCC-4/-9 cells. RESULTS: Progressive increase of Bcl-xL expression and its transcriptional-deregulation was observed with OSCC progression and resistance. Among all the possible upstream regulators of Bcl-xL, such as ERK1/2, Stat-3, p53, AP-1, and NFκB, the TF AP-1 (r = 0.644, p = 0.0001) showed maximum association with Bcl-xL mRNA expression. Though differential expression of AP-1 components were detected in OSCC specimens, with more striking positive-correction of c-Jun (r = 0.381, p = 0.049), c-Fos (r = 0.139, p = 0.488, ns) and Fra-2 (r = 0.664, p = 0.0001) with Bcl-xL expression observed stronger in RCRT tumor subgroup. Further, knockdown of Fra-2 and the application of plant-based phytochemical nimbolide decreased Bcl-xL expression and induced apoptosis in CisR SCC-4/-9 cells. CONCLUSION: Collectively, we have demonstrated the role of Bcl-xL and AP-1 (Fra-2), causing OSCC progression and cisplatin resistance. Targeting Bcl-xL upstream pathway along with the application of nimbolide might be beneficial in eliminating drug-resistant OSCC.

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.

Polymorphisms and haplotypes of TLR4, TLR9 and CYP1A1 genes possibly interfere with high-risk human papillomavirus infection and cervical cancer susceptibility in Jharkhand, India.

BACKGROUND: Expression and single nucleotide polymorphisms (SNPs) of TLR4/9 and CYP1A1 genes are vital for cervical squamous cell carcinoma (CSCC) but considerably vary in different populations. METHODS: A total of 255-subjects from Jharkhand (130-cases, 125-controls) were utilized to obtain the expression/SNP status of TLR4/9, CYP1A1, and E6 (HPV16/18) by RT-PCR, WB, and allele-specific-PCR followed by sequencing. RESULTS: Over-expression of TLR4/9 and high infection of HPV16/18(78.5%) were found to be associated with CSCC. Among the seven SNPs(p1-p7) tested, the CT-genotype (p3:rs1927911; OR = 2.142; p = 0.004) and 'T'-allele (p3; OR = 1.694; p = 0.0061) of TLR4; CC-genotype (p4:rs5743836; OR = 3.307; p = 0.0018), 'C'-allele (p4; OR = 1.895; p = 0.0009), GA-genotype (p5:rs352140; OR = 2.064; p = 0.0172), AA-genotype (p5; OR = 2.602; p = 0.0021) and 'A'-allele (p5; OR = 1.939; p = 0.0002) of TLR9; and the TC-genotype (p6:rs4646903; OR = 1.967; p = 0.0452) and GG-genotype (p7:rs1048943; OR = 2.336; p = 0.0287) and 'G'-allele (p7; OR = 1.685; p = 0.0082) of CYP1A1 were associated with an increased-risk of CSCC. Similarly, the p3:CT-genotype (OR = 1.993; p = 0.0134); p4:CC-genotype (OR = 3.071; p = 0.0057) and 'C'-allele (OR = 1.838; p = 0.0029); p5:AA-genotype (OR = 2.231; p = 0.0147) and 'A'-allele (OR = 1.756; p = 0.0032); p6:TC-genotype (OR = 2.370; p = 0.02); and the p7:GG-genotype (OR = 2.255; p = 0.0488) and 'G'-allele (OR = 1.691; p = 0.0118) showed an association with HPV16/18 infection. Conversely, TLR4 (p1-p2-p3:A-G-T; OR = 3.361; p = 0.029), TLR9 (p4-p5:C-A; OR = 1.786; p = 0.032) and CYP1A1 (p6-p7:C-G; OR = 1.783; p = 0.033) haplotypes with CSCC susceptibility was observed, whereas the TLR4 (p1-p2-p3:A-C-C; OR = 0.4675; p = 8.E-3) and TLR9 (p4-p5:T-G; OR = 0.3937; p = 0.00) haplotypes showed protection against the development of CSCC. Further, though p1:rs10759931 and p2:rs11536889 were found to be insignificant, the p3:CT-genotype, p5:GA/AA-genotype, and p7:GG-genotype were associated with elevated protein; the p4:CC-genotype and p6:TC-genotype were associated with increased mRNA compared to their respective-wild-type-groups. CONCLUSION: The present study revealed an association between TLR4/9 and CYP1A1 polymorphisms with increased HPV16/18 infection susceptibility and CSCC risk among the women of Jharkhand state.

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.

Correction: Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

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.

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.

Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma.

OBJECTIVE: We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. MATERIAL AND METHODS: A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. RESULTS: In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44+ cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. CONCLUSION: The CD44+ /ABCB1+ /ADAM17+ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.

Glycogen synthase kinase-3ß mediated regulation of matrix metalloproteinase-9 and its involvement in oral squamous cell carcinoma progression and invasion.

PURPOSE: Oral squamous cell carcinoma (OSCC)-related deaths mainly result from invasion of the tumor cells into local cervical lymph nodes. It has been reported that progressive basement membrane loss promotes the metastatic and invasive capacities of OSCCs. Matrix metalloproteinase-9 (MMP-9) is known to play a central role in tumor progression and invasion. However, the role of MMP-9 in OSCC invasion has so far remained paradoxical and little is known about its regulation. Here, we aimed to assess MMP-9 expression regulation and its activation by glycogen synthase kinase-3ß during human OSCC progression and invasion. METHODS: In the present study, 178 human OSCC samples, including 118 fresh samples (18 adjacent normal, 42 noninvasive and 58 invasive tumor samples) and 60 archival human tissue microarray (TMA) tongue cancer samples, were included. mRNA expression, protein expression, MMP-9/-2 activity, protein-protein interaction and Snail, c-Myc, ß-catenin and TIMP1 expression were assessed using RT-PCR, immunohistochemistry, Western blotting, co-immunoprecipitation and gelatin zymography analyses, respectively. Wnt5a and LPA mediated MMP-9 regulation was assessed in OCSCC-derived SCC-9 cells exogenously expressing GSK3ß (WT) or non phosphoryable GSK3ß (S9A). RESULTS: We observed a progressive up-regulation/activation of MMP-9 at various stages of oral tumor progression/invasion. Positive correlations were observed between MMP-9 and c-Myc expression, MMP-9 and MMP-2 activity, MMP-9 and TIMP1 expression and MMP-9 activity and TIMP1-MMP-9 interaction. In contrast, a negative correlation between phosphorylated ß-catenin and MMP-9 expression was observed. Conversely, we found that in oral tongue SCC MMP-9 expression was positively correlated with inactivation of GSK3 signaling. Finally, we found that Wnt5a and LPA mediated increased MMP-9 and decreased GSK3ß activities in tongue SCC-derived SCC-9 cells. MMP-9 regulation by GSK3ß was confirmed by using phosphoryable/regulatory GSK3ß (WT construct) and not by non-phosphoryable GSK3ß (S9A construct). CONCLUSIONS: Collectively, our results show that MMP-9 overexpression and activation are important events occurring during OSCC progression/invasion and that this overexpression/activation is regulated by c-Myc, active MMP-2 and inactive GSK3ß mediated pathways.