Hypofractionated Radiation Therapy Suppresses Radioresistance in U87 Human Glioma Cells by Inhibiting Yap1 and Hsp90 Proteins.

BACKGROUND: Radiotherapy plays a vital role in the management of high-grade gliomas. However, the radio resistance of glioma cells limits the effect of radiation and drives recurrence inside the irradiated tumor volume leading to poor outcomes for patients. METHODS: High-grade glioma cell radioresistance significantly contributes to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. An increasing body of evidence complies with the Yes Associated Protein 1 (Yap-1) and heat shock protein 90 (Hsp90) as biomarkers for radioresistance in glioma cells. A number of studies suggest the potential of radioresistance-associated factors as biomarkers and/ or novel therapeutic targets in glioma cells. Thus, it is essential for glioblastoma patients to identify robust druggable targets involved in radioresistance, optimizing irradiation protocol, and understanding their underlying molecular mechanisms. RESULTS: Therefore, in the present study, we hypothesized that hypofractionated Gamma Knife radiation therapy (HF-GKRT) could target Yap-1 and Hsp90 and downregulate the mechanism of radioresistance in high-grade glioma cells. CONCLUSION: For this purpose, expression levels of radioresistance markers Yap-1 and Hsp90 were evaluated after treatment with HF-GKRT, and this was compared with single fraction Gamma Knife radiation therapy (SF-GKRT) in U87MG primary human glioblastoma cell line model. This would help design a novel radiation therapy regimen for glioblastoma patients by reducing the risk of radioresistance.

Identification of Genetic Alterations in Rapid Progressive Glioblastoma by Use of Whole Exome Sequencing.

BACKGROUND: Glioblastoma poses an inevitable threat to patients despite aggressive therapy regimes. It displays a great level of molecular heterogeneity and numerous substitutions in several genes have been documented. Next-generation sequencing techniques have identified various molecular signatures that have led to a better understanding of the molecular pathogenesis of glioblastoma. In this limited study, we sought to identify genetic variants in a small number of rare patients with aggressive glioblastoma. METHODS: Five tumor tissue samples were isolated from four patients with rapidly growing glioblastoma. Genomic DNA was isolated and whole exome sequencing was used to study protein-coding regions. Generated FASTQ files were analyzed and variants were called for each sample. Variants were prioritized with different approaches and functional annotation was applied for the detrimental variants. RESULTS: A total of 49,780 somatic variants were identified in the five glioblastoma samples studied, with the majority as missense substitutions. The top ten genes with the highest number of substitutions were MUC3A, MUC4, MUC6, OR4C5, PDE4DIP, AHNAK2, OR4C3, ZNF806, TTN, and RP1L1. Notably, variant prioritization after annotation indicated that the MTCH2 (Chr11: 47647265 A>G) gene sequence change was putative deleterious in all of the aggressive tumor samples. CONCLUSION: The MTCH2 (Chr11: 47647265 A>G) gene substitution was identified as putative deleterious in highly aggressive glioblastomas, which merits further investigation. Moreover, a high tumor mutation burden was observed, with a signature of the highest substitutions in MUC3A, MUC4, MUC6, OR4C5, PDE4DIP, AHNAK2, OR4C3, ZNF806, TTN, and RP1L1 genes. The findings provide critical, initial data for the further rational design of genetic screening and diagnostic approaches against aggressive glioblastoma.

Gamma Knife Radiosurgery Modulates micro-RNA Levels in Patients with Brain Metastasis.

BACKGROUND: The relation between micro-RNA (miRNA) modulation and immune cell activity in high-dose radiation settings is not clearly understood. OBJECTIVE: To investigate the role of stereotactic radiosurgery (SRS) in (i) the regulation of tumorsuppressor and oncogenic miRNAs as well as (ii) its effect on specific immune cell subsets in patients with metastatic brain tumors (MBT). METHODS: 9 MBT patients who underwent gamma knife-based stereotactic radiosurgery (GKRS) and 8 healthy individuals were included. Serum samples were isolated at three-time intervals (before GKRS, 1 hour, and 1-month post-GKRS). Expressions of tumor-suppressor (miR-124) and oncogenic (miR-21, miR-181a, miR-23a, miR-125b, and miR-17) miRNAs were quantified by qPCR. The lymphocytic frequency (CD3+, CD4+, CD8+, CD56+, CD19+, and CD16+) was investigated by means of flow cytometry. RESULTS: The median age was 64 years (range: 50-73 years). The median prescription dose was 20Gy (range: 16Gy-24Gy), all delivered in a single fraction. The median overall survival and progression- free survival were 7.8 months (range: 1.7-14.9 months) and 6.7 months (range: 1.1-11.5 months), respectively. Compared to healthy controls, baseline levels of oncogenic miRNAs were significantly higher, while tumor-suppressing miRNA levels remained markedly lower in MBT patients prior to GKRS. Following GKRS, there was a reduction in the expression of miR-21, miR-17, and miR-181a; simultaneously, increased expression increased of miR-124 was observed. No significant difference in immune cell subsets was noted post GKRSIn a similar fashion. We noted no correlation between patient characteristics, radiosurgery data, miRNA expression, and immune cell frequency. CONCLUSION: For this specific population with MBT disease, our data suggest that stereotactic radiosurgery may modulate the expression of circulating tumor-suppressor and oncogenic miRNAs, ultimately enhancing key anti-tumoral responses. Further evaluation with larger cohorts is warranted.

Glioma cancer stem cells modulating the local tumor immune environment.

Glioma stem cells (GSCs) drive the resistance mechanism in glioma tumors and mediate the suppression of innate and adaptive immune responses. Here we investigate the expression of mesenchymal-epithelial transition factor (c-Met) and Fas receptor in GSCs and their role in potentiating the tumor-mediated immune suppression through modulation of tumor infiltrating lymphocyte (TIL) population. Tumor tissues were collected from 4 patients who underwent surgery for glioblastoma. GSCs were cultured as neurospheres and evaluated for the co-expression of CD133, c-Met and FasL through flow cytometry. TILs were isolated and evaluated for the lymphocyte subset frequencies including CD3 +, CD4 +, CD8 +, regulatory T cells (FOXP3 + CD25) and microglia (CD11b + CD45) using flow cytometry. Our findings revealed that a significant population of GSCs in all four samples expressed c-Met (89-99%) and FasL (73-97%). A significantly low microglia population was found in local immune cells ranging from 3 to 5%. We did not find a statistically significant correlation between expressions of c-Met + GSC and FasL + GSC with local and systemic immune cells. This may be regarded to the small sample size. The percent c-Met + and FasL + GSC population appeared to be related to percent cytotoxic T cells, regulatory T cells and microglia populations in glioblastoma patients. Further investigation is warranted in a larger sample size.

Physicochemical characterization of C-phycocyanin from Plectonema sp. and elucidation of its bioactive potential through in silico approach.

C-phycocyanin (C-PC), the integral blue-green algae (BGA) constituent has been substantially delineated for its biological attributes. Numerous reports have illustrated differential extraction and purification techniques for C-PC, however, there exists paucity in a broadly accepted process of its isolation. In the present study, we reported a highly selective C-PC purification and characterization method from nontoxic, filamentous and non-heterocystous cyanobacterium Plectonema sp. C-PC was extracted by freeze-thawing, desalted and purified using ion-exchange chromatography. The purity of C-PC along with its concentration was found to be 4.12 and 245 µg/ml respectively.  Comparative characterization of standard and purified C-PC was performed using diverse spectroscopic techniques namely Ultra Violet-visible, fluorescence spectroscopy and Fourier transform infrared (FT-IR). Sharp peaks at 620 nm and 350 nm with UV-visible and FT-IR spectroscopy respectively, confirmed amide I bands at around 1638 cm-1 (C=O stretching) whereas circular dichroism (CD) spectra exhibited α-helix content of secondary structure of standard 80.59% and 84.59% of column purified C-PC. SDS-PAGE exhibited two bands of α and ß subunits 17 and 19 kDa respectively. HPLC evaluation of purified C-PC also indicated a close resemblance of retention peak time (1.465 min, 1.234 min, 1.097 min and 0.905 min) with standard C-PC having retention peak timing of 1.448 min, 1.233 min and 0.925 min. As a cautious approach, the purified C-PC was further lyophilized to extend its shelf life as compared to its liquid isoform. To evaluate the bioactive potential of the purified C-PC in silico approach was attempted. The molecular docking technique was carried out of C-PC as a ligand-protein with free radicals and α-amylase, α-glucosidase, glycogen synthase kinase-3 and glycogen phosphorylase enzymes as receptors to predict the free radical scavenging (antioxidant) and to target antidiabetic property of C-PC. In both receptors free radicals and enzymes, ligand C-PC plays an important role in establishing interactions within the cavity of active sites. These results established the antioxidant potential of C-PC and also give a clue towards its antidiabetic potential warranting further research.

Nanomedicine for glioblastoma: Progress and future prospects.

Glioblastoma is the most aggressive form of brain tumor, accounting for the highest mortality and morbidity rates. Current treatment for patients with glioblastoma includes maximal safe tumor resection followed by radiation therapy with concomitant temozolomide (TMZ) chemotherapy. The addition of TMZ to the conformal radiation therapy has improved the median survival time only from 12 months to 16 months in patients with glioblastoma. Despite these aggressive treatment strategies, patients' prognosis remains poor. This therapeutic failure is primarily attributed to the blood-brain barrier (BBB) that restricts the transport of TMZ from reaching the tumor site. In recent years, nanomedicine has gained considerable attention among researchers and shown promising developments in clinical applications, including the diagnosis, prognosis, and treatment of glioblastoma tumors. This review sheds light on the morphological and physiological complexity of the BBB. It also explains the development of nanomedicine strategies to enhance the permeability of drug molecules across the BBB.

Deciphering the Role of Autophagy in Treatment of Resistance Mechanisms in Glioblastoma.

Autophagy is a process essential for cellular energy consumption, survival, and defense mechanisms. The role of autophagy in several types of human cancers has been explicitly explained; however, the underlying molecular mechanism of autophagy in glioblastoma remains ambiguous. Autophagy is thought to be a "double-edged sword", and its effect on tumorigenesis varies with cell type. On the other hand, autophagy may play a significant role in the resistance mechanisms against various therapies. Therefore, it is of the utmost importance to gain insight into the molecular mechanisms deriving the autophagy-mediated therapeutic resistance and designing improved treatment strategies for glioblastoma. In this review, we discuss autophagy mechanisms, specifically its pro-survival and growth-suppressing mechanisms in glioblastomas. In addition, we try to shed some light on the autophagy-mediated activation of the cellular mechanisms supporting radioresistance and chemoresistance in glioblastoma. This review also highlights autophagy's involvement in glioma stem cell behavior, underlining its role as a potential molecular target for therapeutic interventions.

Targeting Glioblastoma: The Current State of Different Therapeutic Approaches.

BACKGROUND: Glioma is the primary cancer of the central nervous system in adults. Among gliomas, glioblastoma is the most deadly and aggressive form, with an average life span of 1 to 2 years. Despite implementing the rigorous standard care involving maximal surgical removal followed by concomitant radiation and chemotherapy, the patient prognosis remains poor. Due to the infiltrative nature of glioblastoma, chemo- and radio-resistance behavior of these tumors and lack of potent chemotherapeutic drugs, treatment of glioblastoma is still a big challenge. OBJECTIVE: The goal of the present review is to shed some light on the present state of novel strategies, including molecular therapies, immunotherapies, nanotechnology and combination therapies for patients with glioblastoma. METHODS: Peer-reviewed literature was retrieved via Embase, Ovid, PubMed and Google Scholar till the year 2020. CONCLUSION: Insufficient effect of chemotherapies for glioblastoma is more likely because of different drug resistance mechanisms and intrinsically complex pathological characteristics. Therefore, more advancement in various therapeutic approaches such as antitumor immune response, targeting growth regulatory and drug resistance pathways, enhancing drug delivery and drug carrier systems are required in order to establish an effective treatment approach for patients with glioblastoma.

Andrographolide Induces Apoptosis and Cell Cycle Arrest through Inhibition of Aberrant Hedgehog Signaling Pathway in Colon Cancer Cells.

Background Hedgehog signaling pathway (Hh) is abnormally stimulated in colon cancer. Evidence suggests the therapeutic effectiveness of andrographolide against several cancers. This study attempts to delineate the effect of andrographolide on Hh signaling pathway in colon cancer HCT-116 cells. Methods: Effects of andrographolide were studied on HCT-116 cells by evaluating cytotoxicity by MTT assay, morphology assessment, trypan blue exclusion, and colony formation assay; migratory potential by scratch assay; apoptosis by DAPI, Hoechst staining, FITC-Annexin V assay, and caspases activation; mitochondrial membrane potential (ΔΨm) by Mito Tracker and Rhodamine 123. Intracellular ROS by DCFH-DA staining. Cell cycle regulation by flow cytometry. Expression of BAX, BAD, BCL2, Cyclin B1, CDK1, Smo, and Gli1 by qRT-PCR. Interaction between andrographolide and Smo protein by in-silico molecular docking. Results: Andrographolide induced antiproliferative effect on HCT-116 cells in a dose-dependent and time-dependent manner. It also induced apoptosis and anti-migratory effect in HCT-116 cells. In combination with 5FU, andrographolide exhibited synergistic effect. It Induced G2/M phase arrest through downregulating CDK1 and Cyclin B1. Andrographolide also inhibited Hh signaling by downregulating Smo and Gli1 in HCT-116 cells. It showed high affinity toward Smo protein in-silico. Conclusion: Andrographolide repressed the colon cancer cell growth via inhibiting Hh signaling pathway.

Andrographolide Inhibits Proliferation of Colon Cancer SW-480 Cells via Downregulating Notch Signaling Pathway.

BACKGROUND: Recently, the Notch signaling pathway has gained attention as a potential therapeutic target for chemotherapeutic intervention. However, the efficacy of previously known Notch inhibitors in colon cancer is still unclear. The purpose of this study was to investigate the effect of andrographolide on aberrantly activated Notch signaling in SW-480 cells in vitro. METHODS: The cytostatic potential of andrographolide on SW-480 cells was evaluated by 3-(4,5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, morphology assessment, and colony formation assay. The apoptotic activity was evaluated by FITC Annexin V assay, 4',6-diamidino-2-phenylindole (DAPI), Hoechst, Rhodamine 123, and Mito Tracker CMXRos staining. Scratch assay was conducted for migratory potential assessment. 7'-Dichlorodihydrofluorescein Diacetate (DCFH-DA) staining was used to evaluate the Reactive Oxygen Species (ROS) generation. Relative mRNA expression of Bax, Bcl2, NOTCH 1, and JAGGED 1 was estimated by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Cell cycle phase distribution was evaluated by Annexin V-FITC/PI staining. RESULTS: MTT assay demonstrated the dose and time-dependent cytotoxicity of andrographolide on SW-480 cells. It also inhibited the migratory and colony forming potential of SW-480 cells. Furthermore, andrographolide also showed disruption of mitochondrial membrane potential and induced apoptosis through nuclear condensation. Flow cytometric evaluation showed that andrographolide enhanced early and late apoptotic cells and induced upregulation of pro-apoptotic (Bax and Bad) and downregulation of anti-apoptotic Bcl2 in treated SW- 480 cells. Andrographolide augmented intracellular ROS generation and induced G0/G1 phase cell cycle arrest in colon cancer SW-480 cells. Furthermore, andrographolide repressed the Notch signaling by decreasing the expression of NOTCH 1 and JAGGED 1. CONCLUSION: The findings suggested that andrographolide constraint the growth of SW-480 cells through the inhibition of the Notch signaling pathway.

Herbal Medicine for Glioblastoma: Current and Future Prospects.

BACKGROUND: Glioblastoma is one of the most aggressive and devastating tumours of the central nervous system with short survival time. Glioblastoma usually shows fast cell proliferation and invasion of normal brain tissue causing poor prognosis. The present standard of care in patients with glioblastoma includes surgery followed by radiotherapy and temozolomide (TMZ) based chemotherapy. Unfortunately, these approaches are not sufficient to lead a favorable prognosis and survival rates. As the current approaches do not provide a long-term benefit in those patients, new alternative treatments including natural compounds, have drawn attention. Due to their natural origin, they are associated with minimum cellular toxicity towards normal cells and it has become one of the most attractive approaches to treat tumours by natural compounds or phytochemicals. OBJECTIVE: In the present review, the role of natural compounds or phytochemicals in the treatment of glioblastoma describing their efficacy on various aspects of glioblastoma pathophysiology such as cell proliferation, apoptosis, cell cycle regulation, cellular signaling pathways, chemoresistance and their role in combinatorial therapeutic approaches was described. METHODS: Peer-reviewed literature was extracted using Pubmed, EMBASE Ovid and Google Scholar to be reviewed in the present article. CONCLUSION: Preclinical data available in the literature suggest that phytochemicals hold immense potential to be translated into treatment modalities. However, further clinical studies with conclusive results are required to implement phytochemicals in treatment modalities.

Antiproliferative and Apoptotic Properties of Andrographolide Against Human Colon Cancer DLD1 Cell Line.

BACKGROUND: In recent years, natural products have received great attention for cancer prevention owing to their various health benefits, noticeable lack of toxicity and side effects, and the limitations of chemotherapeutic agents. Andrographolide, a labdane diterpenoid is a principal bioactive constituent of Andrographis paniculata Nees, exhibits significant anticancer activity. OBJECTIVE: The efficacy of andrographolide on colon cancer cells is yet to be elucidated completely. Therefore, we investigated the anticancer efficiency of andrographolide in colon cancer DLD1 cell line. METHODS: Antiproliferative activity of andrographolide on DLD1 cells was evaluated by MTT assay, LDH release assay, morphological analysis and colony formation assay. Induction of apoptosis was determined by DAPI staining, Annexin V-FITC staining assay, and caspase-3 activation assay. Role of andrographolide induced cellular reactive oxygen species (ROS) and its association with apoptosis induction in DLD1 cells was elucidated by DCFDA dye. Synergistic ability of andrographolide with 5- fluorouracil (5-FU) and paclitaxel (PTX) was evaluated by MTT assay. RESULTS: Results of the present study indicated that andrographolide declined cell viability of DLD1 cells in a concentration and time-dependent manner. Andrographolide induced apoptosis via nuclear condensation, phosphatidylserine externalization and caspase-3 activation. It also augmented cellular ROS levels which were in turn associated with apoptosis induction in DLD1 cells. Moreover, andrographolide displayed synergistic activity with 5-FU and PTX against DLD1 cells. CONCLUSION: The present study showed that andrographolide demonstrated antiproliferative and apoptotic properties, moreover it also displayed synergistic effect with chemotherapeutic drugs in colon cancer DLD1 cells.