Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma

A 26-Year-Old Female with Systemic Mastocytosis with Associated Myeloid Neoplasm with Eosinophilia and Abnormalities of PDGFRB, t(4;5)(q21;q33)

Various translocations involving the PDGFRB gene are identified in myeloid neoplasms. However, the PRKG2/PDGFRB fusion gene associated with t(4;5)(q21;q33) has previously been reported in only 3 patients. We present the case of a 26-year-old woman with microcytic anemia, basophilia, thrombocytosis, and massive splenomegaly, who was found to have systemic mastocytosis and associated clonal hematological non-mast cell lineage disease (SM-AHNMD), with myeloid neoplasm with PRKG2/PDGFRB rearrangement. Initial findings included basophilia (37%, 4.1 k/μL), hypercellular marrow with eosinophilia, and increased and atypical megakaryocytes, suggestive of myeloproliferative neoplasm. Additional studies revealed large clusters of CD25 positive mast cells, fulfilling the criteria for the diagnosis of systemic mastocytosis. Consistent with prior reports of this translocation, our patient has responded well to imatinib. This case, in conjunction with others in the literature, suggests a possible connection between t(4;5)(q21;q33) PRKG2/PDGFRB and systemic mastocytosis and highlights their favorable response to imatinib

Role of MDR1 C3435T and GABRG2 C588T Gene Polymorphisms in Seizure Occurrence and MDR1 Effect on Anti-Epileptic Drug (Phenytoin) Absorption

Aims: To assess the role of MDR1 and gamma-aminobutyric acid receptor–gamma 2 sub unit (GABRG2) gene polymorphism in seizure susceptibility in generalized seizure (GS) and febrile seizure (FS) patients and to evaluate MDR1 C3435T gene polymorphism’s role in absorption of the anti-epileptic drug, phenytoin (PHT) in a cohort of patients. Methods: One hundred twenty-seven cases of seizure (86 GS and 41 FS) patients were analyzed for MDR1 C3435T and GABRG2 C588T gene polymorphisms using restriction fragment length polymorphismpolymerase chain reaction. Serum PHT levels were analyzed. Results: The T allele of MDR1 C3435T and GABRG2 C588T gene polymorphism was significantly associated with GS in the Indian population (p < 0.05) compared with controls. From the data in GS, CT and TT genotype carriers of the MDR1 gene and TT genotype carriers of the GABRG2 gene had more recurrent seizures compared with others. MDR1 T allele carriers in the seizure reoccurrence (SR) group of GS and FS were high compared with the well-controlled seizure group (with no seizures after treatment). TT genotype carriers in SR group were high in FS (with regard to MDR1 gene polymorphism) and GS (with regard to GABRG2 gene polymorphism) compared with a well-controlled seizure group. MDR1 C3435T gene polymorphism affects serum PHT levels (p < 0.015). Association of dose PHT ratio and genotype groups of MDR1 C3435T gene polymorphism showed a significant association (p < 0.05). MDR1*CC genotype was more common in cases with low serum PHT levels. In addition, it is evident that CT and TT genotype carriers have a high percentage of SR with elevated serum PHT levels. Conclusions: Our results show that the MDR1 3435T and GABRG2 588T alleles play a role in seizure occurrence. Moreover, the MDR1 3435T allele also affects PHT absorption. We suggest MDR1 C3435T and GABRG2 C588T genotyping would be of value in order to lower the risk of concentration-dependent drug toxicity and for better patient management

Efficiency of shRNA plasmid constructs and their effect on adhesion of xenografts to various ECM proteins.

<p>(A) Activity, protein and mRNA expressions of MMP-9, uPAR and cathepsin B in 4910 xenograft cells. MMP-9 and uPA activity was determined by gelatin and fibrin zymography after treatments with scrambled vector (SV-sh), MMP-9 (M-sh), uPAR (U-sh), cathepsin B (C-sh), MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) plasmid shRNAs. Western blot analysis showing MMP-9, uPAR and cathepsin B protein expression levels were reduced after M-sh, U-sh, C-sh, MU-sh and MC-sh treatments. n = 3. RT-PCR of 4910 cells transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh and MC-sh was performed as per standard protocols. Further, quantification of the Western blots (B) revealed significant reductions in protein expressions after M-sh, U-sh, C-sh, MU-sh and MC-sh treatments. n = 3. Values shown are the mean (±SEM). *<i>p</i><0.05 vs. control (C) Adhesion assay was performed to evaluate the effect of SV-sh, M-sh, U-sh, C-sh, MU-sh and MC-sh treatments in 4910 and 5310 glioma xenograft cells on their adhesive potential to collagen (type I), vitronectin, fibronectin and laminin coated plates. Percent adhesion was calculated from the mean obtained from 3 independent experiments and values shown are the mean (±SEM). *<i>p</i><0.05 vs. control.</p

Wound healing, spheroid migration and Matrigel invasion assays.

<p>(A) Reduction in wound healing indicative of reduced migration potential was noticed after treatments with MMP-9 (M-sh), uPAR (U-sh), cathepsin B (C-sh), MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) plasmid shRNAs in both 4910 and 5310 cells. Untreated and SV-sh transfected cells served as controls. (B) Migration of the cells from 4910 and 5310 spheroids transfected with M-sh, U-sh, C-sh, MU-sh and MC-sh was prominently reduced. (C, D) Quantification of wound healing and spheroid migration assays. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments. Similarly, percent migration was calculated from the mean of the average migration obtained from 3 independent experiments. Error bars indicate SEM. *<i>p</i><0.05 vs. control. (E) Matrigel invasion assay of 4910 and 5310 cells transfected with SV-sh, MU-sh and MC-sh. MU-sh and MC-sh treatments prominently reduced the invasive potential of both 4910 and 5310 cells through Matrigel. (F) Invasion was quantified by counting the average number of invaded cells in five different fields with each treatment. Percent invasion was calculated from the mean of the average number of invaded cells obtained from 3 independent experiments. Error bars indicate SEM. *<i>p</i><0.05 vs. control.</p

Involvement of α9β1 integrin on the migration potential of xenograft cells and the effect of bicistronic constructs on the glioma xenograft cell proliferation.

<p>(A) RT-PCR of 5310 and 4910 cells transfected with uPAR (U-fl), MMP-9 (M-fl), and cathepsin B (C-fl) expressing plasmid DNAs was performed as per standard protocols. Prominent increase in respective mRNA expressions noticed after U-fl, M-fl, and C-fl treatments. Further, quantification of the RT-PCR data (B) revealed significant increases in uPAR mRNA and MMP-9 mRNA expression after treatments with M-fl and C-fl, respectively in both the xenograft cells. (C) Wound healing indicative of increased migration potential noticed after treatments with U-fl, M-fl, and C-fl in 5310 cells was reduced with the same treatments in presence of α9β1 antibody. Photographs are the representative images obtained from three independent experiments. (D) Quantification of wound healing assay. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments. Error bars indicate SEM. *<i>p</i><0.05 vs. control. ^#<i>p</i><0.05. (E) FACS analysis showing reduced α9β1 integrin levels in 4910 cells after MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) treatments. (F) Clonogeneic assay depicting the reduced proliferation of 4910 and 5310 cells after MU-sh and MC-sh treatments. After 14 days of incubation, the colonies containing more than 50 cells were counted. Images shown are the representatives obtained from three independent experiments. (G) MTT assay showing significant reduction in the proliferation of 4910 cells from Day 4 to Day 6 after MU-sh and MC-sh treatments. n = 3. *<i>p</i><0.05 vs. control.</p