Practice Patterns and Management Protocols in Trauma across Indian Settings: A Nationwide Cross-sectional Survey.

Background: Trauma is the leading cause of death in India resulting in a significant public health burden. Indian Society of Critical Care Medicine (ISCCM) has established a trauma network committee to understand current practices and identify the gaps and challenges in trauma management in Indian settings. Material and methods: An online survey-based, cross-sectional, descriptive study was conducted with high-priority research questions based on hospital profile, resource availability, and trauma management protocols. Results: Data from 483 centers were analyzed. A significant difference was observed in infrastructure, resource utilization, and management protocols in different types of hospitals and between small and big size hospitals across different tier cities in India (p < 0.05). The advanced trauma life support (ATLS)-trained emergency room (ER) physician had a significant impact on infrastructure organization and trauma management protocols (p < 0.05). On multivariate analysis, the highest impact of ATLS-trained ER physicians was on the use of extended focused assessment with sonography in trauma (eFAST) (2.909 times), followed by hospital trauma code (2.778 times), dedicated trauma team (1.952 times), and following trauma scores (1.651 times). Conclusion: We found that majority of the centers are well equipped with optimal infrastructure, ATLS-trained physician, and management protocols. Still many aspects of trauma management need to be prioritized. There should be proactive involvement at an organizational level to manage trauma patients with a multidisciplinary approach. This survey gives us a deep insight into the current scenario of trauma care and can guide to strengthen across the country. How to cite this article: Sodhi K, Khasne RW, Chanchalani G, Jagathkar G, Kola VR, Mishra M et al. Practice Patterns and Management Protocols in Trauma across Indian Settings: A Nationwide Cross-sectional Survey. Indian J Crit Care Med 2023;27(1):38-51.

An Amino Acid-Based Oral Rehydration Solution Regulates Radiation-Induced Intestinal Barrier Disruption in Mice.

BACKGROUND: Radiotherapy inadvertently affects gastrointestinal (GI) epithelial cells, causing intestinal barrier disruption and increased permeability. OBJECTIVE: We examined the effect of amino acid-based oral rehydration solution (AA-ORS) on radiation-induced changes of intestinal barrier function and epithelial tight junctions (TJs) in a randomized experimental study using a total-body irradiation (TBI) mouse model. METHODS: Eight-week-old male Swiss mice received a single-dose TBI (0, 1, 3, or 5 Gy), and subsequent gastric gavage with AA-ORS (threonine, valine, serine, tyrosine, and aspartic acid) or saline for 2 or 6 d. Intestinal barrier function of mouse ileum was characterized by electrophysiological analysis of conductance, anion selectivity, and paracellular permeability [fluorescein isothiocyanate (FITC)-dextran]. Ultrastructural changes of TJs were evaluated by transmission electron microscopy. Membrane protein and mRNA expression of claudin-1, -2, -3, -5, and -7, occludin, and E-cadherin were analyzed with western blot, qPCR, and immunohistochemistry. Nonparametric tests were used to compare treatment-dose differences for each time point. RESULTS: Saline-treated mice had a higher conductance at doses as low as 3 Gy, and as early as 2 d post-TBI compared with 0 Gy (P < 0.001). Paracellular permeability and dilution potential were increased 6 d after 5 Gy TBI (P < 0.001). Conductance decreased with AA-ORS after 2 d in 3-Gy and 5-Gy mice (P < 0.05 and P < 0.001), and on day 6 after 5 Gy TBI (P < 0.001). Anion selectivity and FITC permeability decreased from 0.73 ± 0.02 to 0.61 ± 0.03 pCl/pNa (P < 0.01) and from 2.7 ± 0.1 × 105 to 2.1 ± 0.1 × 105 RFU (P < 0.001) in 5-Gy mice treated with AA-ORS for 6 d compared with saline. Irradiation-induced ultrastructural changes of TJs characterized by decreased electron density and gap formation improved with AA-ORS. Reduced claudin-1, -3, and -7 membrane expression after TBI recovered with AA-ORS within 6 d, whereas claudin-2 decreased indicating restitution of TJ proteins. CONCLUSIONS: Radiation-induced functional and structural disruption of the intestinal barrier in mice is reversed by AA-ORS rendering AA-ORS a potential treatment option in prospective clinical trials in patients with gastrointestinal barrier dysfunction.

Glucose enhances rotavirus enterotoxin-induced intestinal chloride secretion.

Rotavirus causes severe diarrhea in small children and is typically treated using glucose-containing oral rehydration solutions; however, glucose may have a detrimental impact on these patients, because it increases chloride secretion and presumably water loss. The rotavirus enterotoxin nonstructural protein 4 (NSP4) directly inhibits glucose-mediated sodium absorption. We examined the effects of NSP4 and glucose on sodium and chloride transport in mouse small intestines and Caco-2 cells. Mouse small intestines and Caco-2 cells were incubated with NSP4114-135 in the presence/absence of glucose. Absorption and secretion of sodium and chloride, fluid movement, peak amplitude of intracellular calcium fluorescence, and expression of Ano1 and sodium-glucose cotransporter 1 were assessed. NHE3 activity increased, and chloride secretory activity decreased with age. Net chloride secretion increased, and net sodium absorption decreased in the intestines of 3-week-old mice compared to 8-week-old mice with NSP4. Glucose increased NSP4-stimulated chloride secretion. Glucose increased NSP4-stimulated increase in short-circuit current measurements (I sc) and net chloride secretion. Ano1 cells with siRNA knockdown showed a significant difference in I sc in the presence of NSP4 and glucose without a significant difference in peak calcium fluorescence intracellular when compared to non-silencing (N.S.) cells. The failure of glucose to stimulate significant sodium absorption was likely due to the inhibition of sodium-hydrogen exchange and sodium-glucose cotransport by NSP4. Since glucose enhances intestinal chloride secretion and fails to increase sodium absorption in the presence of NSP4, glucose-based oral rehydration solutions may not be ideal for the management of rotaviral diarrhea.

Pretreatment Immune Status, Predicts Response to Definite Chemo Radiotherapy in Advanced Stages of Cervical Cancer Patients.

Objective: To evaluate the prognostic role of pretreatment CD4 + , CD8 + T lymphocytes in predicting response to definite chemo radiotherapy in advanced cervical cancer. Design: A hospital-based prospective one-year follow-up study. Method: This observational study was conducted on 74 patients with advanced cervical cancer. Pretreatment CD4 + and CD8 + levels in cervical cancer tissue and peripheral blood was noted and quantitatively assessed in patients with complete remission or persistent disease after one year of follow-up. Results: There was a statistically significant association of tumour volume with the remission or persistence of disease. In peripheral blood, mean CD4 + score and CD4 + /CD8 + ratio were significantly higher while mean CD8 + score is significantly lower in patients with remission. Similar results were seen in tumour tissue as well. On Receiver Operating Curve analysis, the cut-off value of CD4 + , CD8 + and CD4 + /CD8 + ratio in predicting remission or persistent disease in peripheral blood was 20.09, 18.51 and 0.41 while in tumor tissue was 19.71, 20.99 and 0.20, respectively. Conclusion: The patients with tumor volume < 100 cm 2 have much higher chances of remission. The patients with higher CD4 + and CD4 + / CD8 + ratio, both in peripheral blood as well as tumor tissue, have higher chances of remission. The cut-off value of CD4 + , CD8 + and CD4 + /CD8 + ratio in predicting remission or persistent disease in peripheral blood was 20.09, 18.51 and 0.41 while in tumor tissue was 19.71, 20.99 and 0.20, respectively.

[Retracted] Oncogenic role of p53 is suppressed by si­RNA bicistronic construct of uPA, uPAR and cathepsin­B in meningiomas both in vitro and in vivo.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 1B, bottom panel, columns 2 and 3; Fig. 4A, top panel, columns 4, 5 and 6, and middle panel, columns 1, 2 and 3; and Fig. 7D, row 1, column 1 and row 2, column 1.
Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused. [the original article was published in International Journal of Oncology 38: 973­983, 2011; DOI: 10.3892/ijo.2011.934]

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[Retracted] α3ß1 integrin promotes radiation-induced migration of meningioma cells.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 2A, right panel, row 3, columns 2, 3 and 4 and Fig. 4D, left panel, row 5, columns 1, 2 and 3; Fig. 4A, row 1, columns 2, 3 and 4, and Fig. 4C, row 1, columns 5, 6 and 7; and Fig. 6C, row 1, column 3, and row 2, column 1.
Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused. [the original article was published in International Journal of Oncology 40: 1615-1624, 2012; DOI: 10.3892/ijo.2011.987].

Optimization studies on floating multiparticulate gastroretentive drug delivery system of famotidine.

The objective of this study was to optimize floating microballoons of famotidine by the emulsion solvent diffusion technique using central composite design. Formulations F1-F15 were prepared using three independent variables (pH of medium, drug: Eudragit S100 ratio and ethanol : dichloromethane ratio) and evaluated for dependent variables (shape, percentage buoyancy, and encapsulation). The optimized formulation F9 was fractionated and a polymer combination of (Eudragit S100 : Eudragit L100-55, 9.5:0.5) resulted in microballoons that exhibited zero order release (94.73%) with 84.20% buoyancy at the end of the eighth hour when studied in the mesh-designed modified USP type II apparatus.

An amino acid-based oral rehydration solution (AA-ORS) enhanced intestinal epithelial proliferation in mice exposed to radiation.

Destruction of clonogenic cells in the crypt following irradiation are thought to cause altered gastrointestinal function. Previously, we found that an amino acid-based oral rehydration solution (AA-ORS) improved gastrointestinal function in irradiated mice. However, the exact mechanisms were unknown. Electrophysiology, immunohistochemistry, qPCR, and Western blot analysis were used to determine that AA-ORS increased proliferation, maturation, and differentiation and improved electrolyte and nutrient absorption in irradiated mice. A single-hit, multi-target crypt survival curve showed a significant increase in crypt progenitors in irradiated mice treated with AA-ORS for six days (8.8 ± 0.4) compared to the saline-treated group (6.1 ± 0.3; P < 0.001) without a change in D0 (4.8 ± 0.1 Gy). The Dq values increased from 8.8 ± 0.4 Gy to 10.5 ± 0.5 Gy with AA-ORS treatment (P < 0.01), indicating an increased radiation tolerance of 1.7 Gy. We also found that AA-ORS treatment (1) increased Lgr5+, without altering Bmi1 positive cells; (2) increased levels of proliferation markers (Ki-67, p-Erk, p-Akt and PCNA); (3) decreased apoptosis markers, such as cleaved caspase-3 and Bcl-2; and (4) increased expression and protein levels of NHE3 and SGLT1 in the brush border membrane. This study shows that AA-ORS increased villus height and improved electrolyte and nutrient absorption.

Caveolin and cavin family members: dual roles in cancer.

Caveolae are specialized plasma membrane subdomains with distinct lipid and protein compositions, which play an essential role in cell physiology through regulation of trafficking and signaling functions. The structure and functions of caveolae have been shown to require the proteins caveolins. Recently, members of the cavin protein family were found to be required, in concert with caveolins, for the formation and function of caveolae. Caveolins have a paradoxical role in the development of cancer formation. They have been involved in both tumor suppression and oncogenesis, depending on tumor type and progress stage. High expression of caveolins and cavins leads to inhibition of cancer-related pathways, such as growth factor signaling pathways. However, certain cancer cells that express caveolins and cavins have been shown to be more aggressive and metastatic because of their increased potential for anchorage-independent growth. Here, we will survey the functional roles of caveolins and of different cavin family members in cancer regulation.

Preparation of cancer-related peptide cocktails that target heterogeneously expressed antigens.

A number of outstanding descriptions of the techniques linked to peptide chain assembly have already been published (Atherton and Sheppard, Solid phase peptide synthesis: a practical approach, pp 55-150, 1989; Stewart and Young, Solid phase peptide synthesis, pp 91-120, 1984; Wellings and Atherton, Methods in enzymology, p 44, 1997). These processes are also described in the operator manuals supplied by the manufacturers of peptide synthesis instrumentation. Accordingly the protocols presented in this chapter are to provide further information on those topics not covered in those publications.

Oncogenic role of p53 is suppressed by si-RNA bicistronic construct of uPA, uPAR and cathepsin-B in meningiomas both in vitro and in vivo.

Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Patients whose tumors recur after surgery and radiation therapy have limited therapeutic options. It has also been reported recently that radiation triggers DNA repair, cell survival and cell proliferation, and reduces apoptosis via the induction of cellular protective mechanisms. Earlier studies have reported that proteases such as uPA, uPAR and cathepsin B play important roles in tumor progression. In the present study, we attempted to determine the effectiveness of two bicistronic siRNA constructs pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation, both in in vitro and in vivo models. Transfection of a plasmid vector expressing double-stranded RNA for uPA, uPAR and cathepsin B significantly induced the sub-G0-G1 cell population by the mitochondrial intrinsic apoptotic pathway. Results showed that pUC efficiently enhanced sub-G0-G1 phases compared to pU2 and was more effective. Interestingly, we observed that in IOMM-Lee cell lines, combined treatment of radiation with pUC and pU2 is more effective in comparison to SF-3061 and MN cell lines. We showed that apoptosis caused by these bicistronic constructs involves Bcl-2, Bcl-xL, p53 inactivation, cytochrome c release from mitochondria and caspase-9 activation, followed by the activation of caspase-3. We also determined that apoptosis caused by pUC and pU2 involves a mechanism which includes inactivation of p53 by its translocation from nucleus to cytoplasm as confirmed by immunofluorescence, which shows the oncogenic potential of p53 in meningiomas. However, the simultaneous RNAi-mediated targeting of uPAR and cathepsin B (pUC), in combination with irradiation, has greater potential application for the treatment of human meningioma in comparison to pU2 by decreasing p53 expression both in vitro and in vivo.

α3ß1 integrin promotes radiation-induced migration of meningioma cells.

Cell motility is influenced by the microenvironment, signal transduction and cytoskeleton rearrangement. Cancer cells become resistant to these control mechanisms and gain the ability to move throughout the body and invade healthy tissues, which leads to metastatic disease. Integrins respond to context-dependent cues and promote cell migration and survival in cancer cells. In the present study, we analyzed the role of integrins in radiation-induced migration of meningioma cells. Migration and cell proliferation assays revealed that radiation treatment (7 Gy) significantly increased migration and decreased proliferation in two cell lines, IOMM-Lee and CH-157-MN. α3 and ß1 integrins were overexpressed at both the protein and transcript levels after radiation treatment and a function-blocking α3ß1 antibody inhibited the radiation-induced migration. Immunofluorescence studies illustrated the localization of α3 integrin and F-actin at the migration front of irradiated cells. Further, an increase in phosphorylation of FAK and ERK was observed, while both FAK phosphorylation inhibitor and FAK shRNA inhibited ERK phosphorylation and downregulated uPA and vinculin. In addition to the co-localization of FAK and ERK at the migration front, these FAK-inhibition results link the downstream effects of ERK to FAK. Correspondingly, U0126 quenched ERK phosphorylation and reduced the expression of molecules involved in migration. Furthermore, brain sections of the animals implanted with tumors followed by radiation treatment showed elevated levels of α3 integrin and active ERK. Taken together, our results show that radiation treatment enhances the migration of meningioma cells with the involvement of α3ß1 integrin-mediated signaling via FAK and ERK.

Chk2-mediated G2/M cell cycle arrest maintains radiation resistance in malignant meningioma cells.

In continuation to our studies on radioresistance in meningioma, here we show that radiation treatment (7Gy) induces G2/M cell cycle arrest in meningioma cells. Phosphorylation of Chk2, Cdc25c and Cdc2 were found to be key events since interference with Chk2 activation and cyclin B1/Cdc2 interaction led to permanent arrest followed by apoptosis. Irradiated cells showed recovery and formed aggressive intracranial tumors with rapid spread and morbidity. Nevertheless, knock down of uPAR with or without radiation induced permanent arrest in G2/M phase and subsequent apoptosis in vitro and in vivo. In conclusion, our data suggest that combination treatment with radiation and uPAR knock down or other inhibitors resulting in non-reversible G2/M arrest may be beneficial in the management of meningiomas.

siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma.

Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.

Downregulation of uPA/uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells.

Hypoxia is known to induce overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) and thus overexpression promotes uPAR-mediated survival signaling in various cancers. Moreover, hypoxia/ overexpression of uPAR in cancer cells promote the epithelial-mesenchymal transition (EMT) and thereby invasiveness and metastasis. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and contributes to EMT, invasion and migration in medulloblastoma cells. Intermittent hypoxia induced expression of mesenchymal markers (i.e., SNAIL, Vimentin and N-cadherin) and reduced expression of epithelial markers (i.e., Zo-1, E-cadherin) in medulloblastoma cells. Further, intermittent hypoxia also leads to enhancement in cell invasion, migration and angiogenesis in medulloblastoma cells. Intermittent hypoxia also inhibited expression of pro-anti-apoptotic proteins (Bax and Bad), and induced expression of anti-pro-apoptotic proteins (Bcl2 and Bcl-xL), and activation of ERK in medulloblastoma cells. Transcriptional inactivation of either uPA or uPAR inhibits the intermittent hypoxia-induced invasion and migration, and expression of Vimentin. uPA/ uPAR downregulation also induces E-cadherin expression and inhibits activation of ERK. Thus, transcriptional inactivation of either uPA or uPAR enhances the apoptotic response in medulloblastoma cells exposed to intermittent hypoxia. This study provides evidence of the anti-tumor efficacy of down-regulation of uPA or uPAR in medulloblastoma tumors to target hypoxia-induced cell EMT, invasion and migration, to achieve better therapeutic outcomes in the treatment of malignant medulloblastoma.

Suppression of uPA and uPAR blocks radiation-induced MCP-1 mediated recruitment of endothelial cells in meningioma.

Chemokines play a vital role in recruiting various cell types in the process of tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response that might lead to the expression of several chemokines. In the present study, we investigated the mechanism of monocyte chemoattractant protein-1 (MCP-1) induction by radiation in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). After radiation, meningioma cell lines (IOMM Lee and SF-3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell conditioned medium (CM) showed an increased ability to attract HMEC and to stimulate MCP-1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC. This chemotactic activity and angiogenic stimulator effect on HMEC were almost abrogated by depleting MCP-1 from the irradiated cancer cell CM. Further, inhibition of either ERK activation/expression or NF-κB nuclear translocation hindered radiation-induced MCP-1 expression in both meningioma cell lines. Further, supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR, simultaneously by transfecting the cancer cells with bi-cistronic siRNA-expressing plasmid (pU) inhibited radiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. In addition, pU-transfected cancer cells (with or without radiation) reduced radiation-induced MCP-1 and blocked the recruitment of other cell types during the inflammatory process induced by radiation both in in vitro and in vivo conditions.

uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

BACKGROUND: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. CONCLUSION/SIGNIFICANCE: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

Radiation-inducible silencing of uPA and uPAR in vitro and in vivo in meningioma.

Stereospecific radiation treatment offers a distinct opportunity for temporal and spatial regulation of gene expression at tumor sites by means of inducible promoters. To this end, a plasmid, pCArG-U2, was constructed by incorporating nine CArG elements (in tandem) of EGR1 gene upstream to uPA and uPAR siRNA oligonucleotides in a pCi-neo vector. Radiation-induced siRNA expression was detected in a meningioma cell line (IOMM-Lee). Immunoblotting and RT-PCR analyses confirmed downregulation of uPA and uPAR. A similar effect was observed in transfected cells followed by H2O2 treatment. Moreover, pre-treatment of transfected cells with N-acetyl L-cysteine blocked the silencing of uPA and uPAR, which further confirmed the oxidative damage-mediated downregulation. Cell proliferation assays and Western blot analysis for apoptotic molecules confirmed cell death in a radiation-inducible fashion. Migration and matrigel invasion assays also revealed a marked decrease in migration and invasion. Immunocytochemistry showed a marked decrease in uPA and uPAR levels in transfected and irradiated cells. H&E staining revealed a decrease in the pre-established tumor volume among the animals treated with pCArG-U2 and radiation. Immunohistochemistry of the brain sections established with intracranial tumors also revealed a marked decrease in uPA and uPAR in a radiation-inducible fashion. Taken together, our data suggest pCArG-U2 as a suitable candidate for radiation-inducible gene therapy.