Prajna Mishra

Mixed Lineage Kinase-3/JNK1 Axis Promotes Migration of Human Gastric Cancer Cells following Gastrin Stimulation

Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

Caspase-mediated Cleavage of β-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells

A delicate balance between cell death and survival pathways maintains normal physiology, which is altered in many cancers, shifting the balance toward increased survival. Several studies have established a close connection between the Wnt/β-catenin pathway and tumorigenesis, aberrant activation of which might contribute toward increased cancer cell growth and survival. Extensive research is underway to identify therapeutic agents that can induce apoptosis specifically in cancer cells with minimal collateral damage to normal cells. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis specifically in tumor cells, many cancer cells develop resistance, which can be overcome by combinatorial treatment with other agents: for example, peroxisome proliferator-activated receptorγ (PPARγ) ligands. To identify the molecular target mediating combinatorial drug-induced apoptosis, we focused on β-catenin, a protein implicated in oncogenesis. Our results show that co-treatment of TRAIL-resistant cancer cells with TRAIL and the PPARγ ligand troglitazone leads to a reduction ofβ-catenin expression, coinciding with maximal apoptosis. Modulation of β-catenin levels via ectopic overexpression or small interference RNA-mediated gene silencing modulates drug-induced apoptosis, indicating involvement of β-catenin in regulating this pathway. More in-depth studies indicated a post-translational mechanism, independent of glycogen synthase kinase-3β activity regulating β-catenin expression following combinatorial drug treatment. Furthermore, TRAIL- and troglitazone-induced apoptosis was preceded by a cleavage of β-catenin, which was complete in a fully apoptotic population, and was mediated by caspases-3 and -8. These results demonstrate β-catenin as a promising new target of drug-induced apoptosis, which can be targeted to sensitize apoptosis-resistant cancer cells.

Mixed Lineage Kinase 3 Modulates β-Catenin Signaling in Cancer Cells

Background: β-Catenin mediates a wide variety of cellular processes, but the signaling pathways regulating β-catenin downstream events are not fully understood. The role of MLK3 in modulating β-catenin pathway has not been reported earlier. Results: MLK3 can induce β-catenin stabilization but inhibit conventional β-catenin/TCF transcriptional activation. Conclusion: These provide a new mechanism of regulating β-catenin/TCF axis. Significance: MLK3 can be targeted in regulating the growth of β-catenin overexpressing tumors. Expression of β-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3β (GSK3β)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/β-catenin signaling or mutations of members of the degradation complex) can result in β-catenin stabilization and activation of β-catenin/T-cell factor (TCF) signaling. β-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control β-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between β-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce β-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with β-catenin and phosphorylate it in vitro. Overexpression of GSK3β-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3β pathway. Surprisingly, despite stabilizing β-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A β-catenin. These resulted in reduced expression of β-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease β-catenin/TCF interaction but promoted interaction between β-catenin and KLF4, a known repressor of β-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and β-catenin resulted in significant G2/M arrest. These studies provide a novel insight toward the regulation of β-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of β-catenin signaling.

Reversion-inducing cysteine-rich protein with Kazal motifs and its regulation by glycogen synthase kinase 3 signaling in oral cancer

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and glycogen synthase kinase (GSK3) are novel tumor suppressors, and emerging evidence has suggested their active role in oral cancer pathogenesis. In the present study, 112 human samples, including 55 fresh samples of 14 adjacent normal tissues, 25 noninvasive oral tumors, and 18 invasive tumors, were included. The messenger RNA (mRNA) expression, protein expression, and promoter methylation of the RECK gene, as well as the expression of GSK3β, phospho/total β-catenin, and c-myc, were measured by RT-PCR, bisulphate modification-PCR, immunohistochemistry, and Western blot analysis. Additionally, ectopic expression of in/active GSK3β was performed in cell culture experiments. This study provided information on the progressive silencing of RECK gene expression at the protein and mRNA levels paralleled with promoter hypermethylation at various stages of oral tumor invasion. RECK expression and the hypermethylation of the RECK gene promoter were negatively and positively correlated with pS9GSK3β/c-myc expression, respectively. Further, a negative trend of RECK protein expression with nuclear β-catenin expression was observed. Induced expression of active GSK3β reversed the RECK silencing in SCC9 cells. Collectively, our results demonstrated that the silencing of the RECK gene, possibly regulated by the GSK3β pathway, is an important event in oral cancer invasion and this pathway could be exploited for therapeutic interventions.

Co-delivery of docetaxel and doxorubicin using biodegradable PEG-PLA micelles for treatment of breast cancer with synergistic anti-tumour effects

ABSTRACT Poly(ethylene glycol)-poly(lactic acid) copolymer, prepared by ring opening polymerization, was used as a single platform to co-deliver both hydrophilic doxorubicin and hydrophobic docetaxel (DTX) in a simulated physiological environment. The average size of the negatively charged drug loaded polymeric micelles were found to be 293 nm. The drug loading (%) and encapsulation efficiency (%) were calculated to be 1.21 and 59.0, respectively. The in vitro cytotoxicity test using MCF7 breast cancer cells was conducted using 1 × 104 cells in 10% FBS and 1% antibiotic, and the absorbance of formazan was evaluated at 570 nm. Cell growth inhibition by MTT assay showed viability of 33% of the MCF7 cells after treatment with drug-loaded micelles for 48 h. Controlled release of drugs from the polymeric micelles indicated a burst release effect initially; whereas, 98% of drug could be released at pH 7.4 within a time period of 96 h. Time period for drug release shorten to 48 h only in simulated mild acidic pH (5.4) condition. The in vitro drug release study from micelles indicated synergistic cytotoxicity effect in human metastatic breast cancer MCF7 cell.

Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3β 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, GSK3β, 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 GSK3β 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/GSK3β 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/GSK3β activation and driving tumor invasion/migration.

Development of docetaxel-loaded PEG–PLA nanoparticles using surfactant-free method for controlled release studies

ABSTRACT Docetaxel (DTX)-loaded poly(ethylene glycol)–poly(D,L-lactide) is prepared by nanoprecipitation method in the absence of any surfactants. The average particle size of the copolymer was found to be 101 nm. The drug entrapment efficiency (%) and drug loading (%) of polymer were found to be 9.471 ± 0.047 and 94.71 ± 0.466, respectively. The in vitro drug release characteristics show the controlled release of 98% of docetaxel in 72 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis measured in terms of cleaved Poly(ADP-ribose) Polymerase (PARP) and cleaved caspase-3 protein expression shows that the copolymer has better cytotoxicity effect and apoptosis in comparison to free DTX in HeLa cells. GRAPHICAL ABSTRACT