Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma

Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date.In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling proteins in glioma cells both in vitro and in vivo.We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas

Inhibition of cell proliferation by hUCBSC treatments.

<p>(A) Control and hUCBSC-treated glioma cells at different time points were analyzed for cell proliferation by incorporation of BrdU. *Significant at <i>p</i><0.05 compared to respective control cells. **Significant at <i>p</i><0.01 compared to respective control cells (by <i>t</i>-test). (B) Control, EGF-treated and EGF+hUCBSC treated glioma cells were analyzed for cell proliferation. *Significant at <i>p</i><0.05 both for control <i>vs.</i> EGF+hUCBSC treatment and EGF <i>vs.</i> EGF+hUCBSC treatments (one-way ANOVA followed by Bonferroni's post hoc test). (C) Control, TMZ treated, TMZ+hUCBSC treated glioma cells were subjected to cell proliferation analysis by BrdU incorporation. *Significant at <i>p</i><0.05; **Significant at <i>p</i><0.01 both for control <i>vs.</i> TMZ treatment and control <i>vs.</i> TMZ+hUCBSC treatments (one-way ANOVA followed by Bonferroni's post hoc test). For all these tests, raw data was taken into consideration to perform statistical analyses and then results were presented as % control values. Each bar represents n≥6.</p

Downregulation of pEGFR and phospho-c-Src in mitochondria in hUCBSC treatments.

<p>(A) Control and hUCBSC-treated U251, U87 and 5310 cells were labeled with pEGFR and Mito Tracker green and processed for immunofluorescence. (B) Control and hUCBSC-treated U251 and 5310 tissue sections were labeled with pEGFR and Mito Tracker green and processed for immunofluorescence. pEGFR was conjugated with Alexa Fluor 594 (red) secondary antibody (n≥3; bar = 100 µm). (C) Control and hUCBSC-treated U251, U87 and 5310 cells were labeled with phospho-c-Src and Mito Tracker green and processed for immunofluorescence. (D) Control and hUCBSC-treated U251 and 5310 tissue sections were labeled with phospho-c-Src and Mito Tracker green and processed for immunofluorescence. Phospho-c-Src was conjugated with Alexa Fluor 594 (red) secondary antibody (n≥3; bar = 100 µm).</p

Expression of EGFR in GBM-patient derived specimens.

<p>(A) Control brain and GBM-patient derived specimens were subjected to DAB immunohistochemistry using anti-rabbit EGFR antibody (bar = 200 µm). (B) mRNA extracted from the above specimens was subjected to RT-PCR using primers specific for EGFR, FAK, c-Src and β-actin (loading control). (C) Quantitative analysis of (B). (D) Tissue lysates (40 µg protein) of the above specimens were subjected to Western blotting using the following antibodies: EGFR, pEGFR, FAK, pFAK, c-Src and phospho-c-Src. Mouse anti-GAPDH (1∶1000) served as the loading control. (E) Quantitative analysis of (D). *Significant at <i>p</i><0.05 compared to control brain samples.</p

Inhibition of EGFR by hUCBSC treatment.

<p>For <i>in vitro</i> experiments, glioma cells (U251, U87 and 5310) were co-cultured with hUCBSC for 72 hours. For <i>in vivo</i> experiments, U251 (1×10⁶ cells) and 5310 (8×10⁵ cells) tumor cells were intracerebrally injected into the right side of the brain of the nude mice. Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with 2×10⁵ hUCBSC by intracranial injection. Seven days after tumor implantation, the mice were injected with hUCBSC towards the left side of the brain. Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with appropriate antibodies. (A) U87, U251 and 5310 glioma cells alone and after co-culture with hUCBSC were labeled with mouse anti-EGFR antibody and processed for immunofluorescence. EGFR was conjugated with Alexa Fluor 488 (green) secondary antibody (n = 3; bar = 100 µm). (B) Control and hUCBSC-treated tumor sections were labeled with mouse anti-EGFR antibody and processed for DAB immunohistochemistry. For (C) and (E), total mRNA was extracted, converted to cDNA, and subjected to semi-quantitative RT-PCR. (C) Control and hUCBSC-treated U251, U87 and 5310 <i>in vitro</i> cDNAs were subjected to RT-PCR using EGFR, FAK and c-Src primers. (D) Quantitative analysis of (C). *Significant at <i>p</i><0.05 compared to respective control glioma cells. **Significant at <i>p</i><0.01 compared to respective control glioma cells. (E) Control and hUCBSC-treated U251 and 5310 <i>in vivo</i> cDNAs were subjected to RT-PCR using EGFR, FAK and c-Src primers. β-actin served as a loading control. (F) Quantitative analysis of (E). *Significant at <i>p</i><0.05 compared to respective control glioma tissues. For (G) and (I), 40 µg of proteins were loaded onto 8% gels and transferred onto nitrocellulose membranes, probed with respective antibodies, and developed by autoradiography. (G) Control and hUCBSC-treated U251, U87 and 5310 glioma cell lysates were processed and immunoblotted with EGFR, FAK and c-Src antibodies. (H) Quantitative analysis of (G). **Significant at <i>p</i><0.01, *Significant at <i>p</i><0.05 compared to respective control glioma cells. (I) Control and hUCBSC-treated U251 and 5310 <i>in vivo</i> brain tissue lysates were processed by standard Western blotting and probed with EGFR, FAK and c-Src antibodies. GAPDH served as a loading control. (J) Quantitative analysis of (I). **Significant at <i>p</i><0.01, *Significant at <i>p</i><0.05 compared to respective control glioma tissue lysates. For (C–J), n≥3.</p

Downregulation of EGFR-mediated signaling molecules in hUCBSC treatments.

<p>For (A), (C), (E), (G) and (I) 40 µg of proteins were loaded onto 8–12% gels and transferred onto nitrocellulose membranes, probed with respective antibodies, and developed by autoradiography. (A) <i>In vitro</i> samples probed with pEGFR, pFAK, p-c-Src and GAPDH. (B) Quantitative analysis of (A). *Significant at <i>p</i><0.05 compared to respective control glioma cells. (C) <i>In vivo</i> cytosolic fractions probed with pEGFR, pFAK, p-c-Src and GAPDH. n = 3. (D) Quantitative analysis of (C). *Significant at <i>p</i><0.05 compared to respective control glioma tissues. (E) <i>In vivo</i> mitochondrial fractions probed with pEGFR, pFAK, p-c-Src and Cox IV. n = 3. (F) Quantitative analysis of (E). *Significant at <i>p</i><0.05 compared to respective control glioma tissues. Control and hUCBSC-treated U251, U87 and 5310 glioma cell lysates (G) and control and hUCBSC-treated U251 and 5310 glioma tissue lysates (I) were processed for immunoblotting and probed with MEK1, RAF1, Stat3 and PI3K antibodies. GAPDH served as a loading control. (H) Quantitative estimation of (G). *Significant at <i>p</i><0.05 compared to respective glioma control cells. **Significant at <i>p</i><0.01 compared to respective glioma control cells. (J) Quantitative estimation of (I). *Significant at <i>p</i><0.05 compared to respective glioma control tissues. **Significant at <i>p</i><0.01 compared to respective glioma control tissues. (K) Tumor size volume estimated in control and hUCBSC treated nude mice brains. *Significant at <i>p</i><0.05 compared to glioma controls. Error bars indicate ± SD. For (A) to (K), n≥3.</p

Colocalization of pEGFR and pFAK in clinical samples.

<p>Control brain and GBM-patient derived specimens were labeled with pEGFR and pFAK antibodies along with Mito Tracker (green) and processed for immunofluorescence. pEGFR was conjugated with Alexa Fluor 350 (blue) and pFAK was conjugated with Alexa Fluor 594 (red) secondary antibodies (bar = 100 µm).</p

Assessment of internet addiction during COVID 19 pandemic in India, 2022

Introduction: The effect of COVID-19-related changes (such as disturbance of face-to-face human association, breakdown of propensities, vulnerability almost long-term, money related frailty, and depression) on people's mental well-being.&nbsp; The enormous utilize of the Web can be a double-edged sword: on the one hand, get to to innovation has cultivated people's well-being and has encouraged the get to to social back; on the other hand, it may have turned into tricky utilize, particularly with respect to compulsive utilize and cognitive distraction. &nbsp;Methods: A sample of 311 was calculated by using the Raosoft software. Descriptive analysis, univariate analysis and multivariate analysis were carried out. Binary logistic regression and multiple logistic regression were used for analysis to access the associated factors with internet addiction. Results: The majority of respondents 323(61.5%) belonged to the normal level of internet addiction, which was followed by Moderate level of internet addiction by 113(21.5%) respondents. 8(1.5%) respondents were severely addicted to internet. Conclusion: During COVID 19 pandemic, the prevalence of psychological problems such as Internet Addiction increased especially among young adults. Therefore, national policy should be refined to reduce the stressful lifestyle in order to reduce the impact of internet addiction especially for young adults in India