Habb-e-Asgandh Suppresses Cell Proliferation and Induces Apoptosis through Mitochondria Dysfunction in Multiple Myeloma Cells (RPMI8226).

OBJECTIVE: This study was conducted to assess the anti-neoplastic properties of Habb-e-Asgandh in multiple myeloma cells (RPMI8226). METHODS: Multiple myeloma cells (RPMI8226) were cultured according to the ATCC's instruction. The anti-proliferative effect of HeA was assessed by MTT assay and proliferating cellnuclear antigen (PCNA) activity. Cell cycle analysis, cellular apoptosis, and mitochondria membrane potential analysis was done by flow cytometry. Total antioxidants, migratory potential, angiogenesis and inflammatory biomarkers were also estimated after treatment of RPMI8226 with HeA. RESULTS: LD30 and LD50 dose of HeA was 0.3mg/ml and 0.5mg/ml respectively determined by MTT assay and also confirmed by a reduced PCNA activity. Cell cycle analysis of RPMI8226 cells revealed that sub-G0/G1 phase increases upon treatment with HeA alone or in combination with lenalidomide. Annexin V-FITC/PI is used to detect early apoptosis, late apoptosis and necrotic cells and results showed that percentage of apoptotic cells increased in RPMI8226 cells after treatment with HeA. Also, HeA induces loss of mitochondria membrane potential (MMP) in MM cells in-vitro as measured by cationic JC1 dye staining. Upon treatment, the abnormal overexpression of oncogenic protein, AKT serine/threonine kinase has also been reduced. Furthermore, anti-oxidants level also increased while migratory potential, angiogenesis and inflammation decreased in multiple myeloma cell line upon treatment with HeA. CONCLUSION: Collectively, our results demonstrated that integrative therapy of habb-e-asgandh efficiently eliminates the need to use higher dose of lenalidomide for multiple myeloma treatment.

Itrifal-e-Aftimoon potentiates imatinib-induced anti-leukemic effect by influencing FAK/STAT/Akt/ERK signalling pathways against chronic myeloid leukaemia in vitro.

OBJECTIVES: Limited treatment options are available for advanced stages of chronic myeloid leukaemia (CML). Moreover, patients' relapse after a short remission period, which prompts them to identify a potent drug with the least toxicity. An Unani herbal formulation, Itrifal-e-Aftimoon (IEA) is used for certain neurological disorders, however, its antitumor potential has not been reported yet in any malignancy, including CML. METHODS: The aqueous extract of IEA was characterized by HPLC/LC-MS and used alone or in combination with standard drug, imatinib in CML cell lines (K562, KU812) in vitro to assess its effect on cancer-associated parameters such as cytotoxicity, cell cycle, apoptosis, oxidative stress, inflammation, angiogenesis, and certain signalling pathways. RESULTS: LC-MS characterization of IEA showed the presence of antitumor compounds including catechin and caffeic acid. Treatment with IEA caused cytotoxicity and arrested cells in the sub-G0/G1 phase. Subsequent assays confirmed apoptosis-mediated cell death with mitochondrial membrane depolarization and alleviation of oxidative stress. IEA abrogates IL-6, VEGF, angiopoietin-2, and alters Th1/Th2 cytokines. IEA potentiated the effect of imatinib even at lower doses by affecting FAK/STAT/Akt/ERK pathways. CONCLUSION: IEA possesses antitumor potential against CML and increases the efficacy of imatinib when used in combination, suggesting utilization of IEA as an adjuvant therapy for better management of CML in the future.

Unveiling molecular associations of polymorphic variants of VDR gene (FokI, BsmI and ApaI) in multiple myeloma patients of Indian population.

Multiple myeloma (MM) is a plasma cell malignancy frequently accompanied with skeletal co-morbidity. Vitamin D (1,25(OH)2D) is an important mediator of skeletal homeostasis that mediates its effect by binding to vitamin D receptor (VDR), a steroid family receptor and modulates various downstream pathways. Multiple polymorphisms have been determined in VDR gene that witnessed significant association with cancer development and progression. Therefore, in this maiden study, we recruited 75 newly diagnosed MM patients and 75 control subjects. 25-hydroxy vitamin D (25(OH)D) levels were measured in all recruited study subjects. Further, PCR-RFLP was performed in DNA samples of recruited study subjects. Results demonstrated significantly decreased 25(OH)D levels in MM patients compared to controls. Additionally, decreased 25(OH)D levels in MM patients inversely associated with disease severity. Further, single nucleotide polymorphism (SNP) analysis of VDR gene showed significantly higher risk of MM disease development in Ff + ff, Aa + aa, and Bb + bb genotypes. Additionally, FokI f, ApaI a and BsmI b alleles were significantly associated with MM occurrence. In conclusion, this study provided initial evidences of association between 25(OH)D insufficiency, VDR gene polymorphism and MM development. Thus, we suggest that a study involving assessment of 25(OH)D levels and VDR gene polymorphism in large patients' cohort might substantiate their role in MM development which would further provide impetus to give 25(OH)D supplementation along with conventional chemotherapeutic agents for myeloma treatment in future.

RASSF1A-Hippo pathway link in patients with urothelial carcinoma of bladder: plausible therapeutic target.

RASSF1A is a tumor suppressor gene, and its hypermethylation has been observed in cancers. RASSF1A acts as an upstream regulator of Hippo pathway and modulates its function. The aim of this study was to analyze expression of RASSF1A, Hippo pathway molecules (YAP, MST) and downstream targets (CTGF, Cyr61 and AREG) in bladder cancer patients. Later, the link between RASSF1A and Hippo pathway and a potential therapeutic scope of this link in UBC were also studied. MSPCR was performed to study methylation of RASSF1A promoter. Expression of molecules was studied using qPCR, Western blot and IHC. The link between RASSF1A and Hippo pathway was studied using Spearman's correlation in patients and validated by overexpressing RASSF1A in HT1376 cells and its effect on Hippo pathway was observed using qPCR and Western blot. Further therapeutic potential of this link was studied using MTT and PI assays. The expression of RASSF1A was lower, whereas the expression of YAP, CTGF and CYR61 was higher. The expression of RASSF1A protein gradually decreased, while the expression of YAP, CTGF and CYR61 increased with severity of disease. Based on Spearman's correlation, RASSF1A showed a negative correlation with YAP, CTGF and CYR61. YAP showed a positive correlation with CTGF and CYR61. To validate this link, RASSF1A was overexpressed in HT1376 cells. Overexpressed RASSF1A activated Hippo pathway, followed by a decrease in CTGF and CYR61 at mRNA, and enhanced cytotoxicity to chemotherapeutic drugs. This study finds a previously unrecognized role of RASSF1A in the regulation of CTGF and CYR61 through mediation of Hippo pathway in UBC and supports the significance of this link as a potential therapeutic target for UBC.

Role of Trace Elements, Oxidative Stress and Immune System: a Triad in Premature Ovarian Failure.

The risk of premature ovarian failure (POF) increases in association with alteration in immunological parameters and oxidative stress (OS). Adequate intake of trace elements is required for antioxidant property and immune defense mechanism. The aim of this study was to explore the involvement of trace elements, OS, and immunological parameters in POF. This was a cross-sectional, case-control study, involving 65 participants divided into the POF (n = 35) and control (n = 30) groups. Serum levels of Se, Zn, and Cu were determined along with hormonal, OS, and immunological markers. POF group had significantly lower levels of Zn, Cu, Se, and Zn:Cu ratio. However, Se:Cu ratio was not significant between the groups. FSH and LH levels were negatively correlated with Zn and Cu levels and positively correlated with Se levels. Estrogen levels were negatively correlated with all the studied trace elements. Inter-element association between Zn and Se was significant in POF (r = - 0.39, p = 0.02) compared to control group (r = - 0.078, p = 0.65). In all the POF patients, SOD and GPx activities were significantly (p < 0.05) lower and MDA level was higher (p > 0.05) than control group. B cell marker CD19 was significantly (p < 0.0001) high in POF group. There are involvement of trace elements in hormonal regulation and antioxidant defense mechanism, which once gets altered leads to high ROS generation and affect functions of the immune system. Exaggereative immune system causing higher expression of B cell associated markers (CD19) leading to autoimmune condition in POF.

Genetic endowment of proinflammatory cytokine tumor necrosis factor-α (-) 308 G > A polymorphism to Parthenium hysterophorus-induced airborne contact dermatitis in an Indian cohort.

BACKGROUND: Parthenium dermatitis is a common airborne allergic health problem that induces a cell-mediated hypersensitivity immune response involving activated T lymphocytes, which culminates in injury to the skin. The disease is manifested as itchy erythematous papules and plaques and primarily affects the exposed areas and flexures. This study aimed to identify the role of tumor necrosis factor (TNF)-α (-) 308 G>A polymorphism in the pathogenesis of parthenium dermatitis. MATERIALS AND METHODS: A total of 120 subjects, including 60 patients exclusively diagnosed for parthenium dermatitis and 60 healthy individuals, were included in the study. The genotyping of the TNF-α (-) 308 G>A region was carried out by the amplification refractory mutational system. RESULTS: In the present study, we demonstrated that polymorphism of the TNF-α (-) 308 position (A and/or G) was not statistically significant, and there was no difference in the distribution of any alleles of this locus in cases and controls. CONCLUSION: The present study suggests that there is a lack of association of potent proinflammatory cytokine TNF-α (-) 308 G>A polymorphism in parthenium dermatitis in the Indian cohort. It interprets genetically endowed transcriptional capacity due to this particular single nucleotide polymorphism but does not support the prevalence of high serum levels of TNF-α in parthenium-induced skin allergic inflammation.

Dysregulation of T(H) type cytokines in the patients of Parthenium induced contact dermatitis.

BACKGROUND: Parthenium contact dermatitis is a major health problem caused by a cosmopolitan weed Parthenium hysterophorus. It is a T cell-mediated immune injury and disease manifests as itchy erythematous papules, papulovesicular and plaque lesions on exposed areas of the body. We studied the involvement of T(H)1/T(H)2/T(H)17/Treg type responses by assaying various cytokines in Parthenium dermatitis. METHODS: The study includes 50 patients of Parthenium dermatitis confirmed by patch testing and 50 healthy subjects. The serum levels of T(H)1, T(H)2, T(H)17 and Treg cytokines were estimated by high sensitivity sandwich ELISA and were compared statistically between groups using ANOVA. RESULTS: The mean concentration of T(H)1 cytokines (p<0.001) and IL-17 (p<0.001) were increased significantly as compared to controls. In contrast, decrease in levels of IL-10 (p<0.002) and TGF-ß (p<0.001) were significant and levels of IL-4 (p<0.262) were insignificant whereas no alterations in the total IgE concentrations (p<0.976) was observed. CONCLUSION: The induction of T(H)1 and T(H)17 cytokines reinforce the need of detailed analysis of immune dysregulation in Parthenium dermatitis and might add some insight in the pathogenesis, diagnosis and current treatment modalities of this disease.

Study of pro- and anti-inflammatory cytokine profile in the patients with parthenium dermatitis.

BACKGROUND: Parthenium dermatitis is a common airborne allergic contact dermatitis induced by exposures to the weed Parthenium hysterophorus. The disease manifests as itchy erythematous papules, papulovesicular and plaque lesions on exposed areas of the body. OBJECTIVES: The aim of this study was to show the alterations in pro/anti-inflammatory cytokines in parthenium dermatitis. METHODS: The study included 50 patients with parthenium dermatitis confirmed by patch testing using aqueous extracts of P. hysterophorus and 50 age-matched healthy controls. The levels of pro-inflammatory [tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-17] and anti-inflammatory (IL-4 and IL-10) cytokines were estimated by commercially available high sensitivity enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: All the dermatitis patients showed significantly (P < 0.001) elevated levels of TNF-α, IL-6, IL-8, and IL-17 levels as compared to healthy controls. In contrast, the anti-inflammatory cytokine IL-4 showed an insignificant decrease (P < 0.217) and a decrease in level of IL-10 was statistically significant (0.001) compared with controls. CONCLUSIONS: The present study suggests the involvement of pro-inflammatory cytokines in the pathogenesis of parthenium dermatitis. A decrease in levels of anti-inflammatory cytokines was demonstrated, which could not downregulate pro-inflammatory cytokines in parthenium dermatitis.

Altered antioxidant status and lipid peroxidation in Indian patients with urothelial bladder carcinoma.

OBJECTIVES: Urothelial carcinoma of bladder is the second most common urological malignancy after prostate cancer. Recently, there has been increased interest in research of the role of free radicals and antioxidant materials in the prevention, treatment, and alleviation of therapy-related side effects of cancer. In the present study, we aimed to assess the alterations in the levels of antioxidant vitamins, activities of defense enzymes, circulating lipid peroxide, and total antioxidant activity (AOA) in patients with urothelial carcinoma of bladder and correlate these changes with the grade and severity of the disease. MATERIALS AND METHODS: The study cohort consisted of 90 subjects; 50 patients with bladder UC (25, low grade; 10, high grade; 15, muscle invasive) and 40 healthy controls. Vitamins C and E, malondialdehyde (MDA), and AOA were estimated using standard protocols. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assayed using commercially available kits. RESULTS: The serum levels of vitamins C and E, whole blood levels of SOD and GPx, and serum AOA was significantly lower (P < 0.001) while serum MDA levels were significantly higher (P < 0.001) in patients than in controls, indicating presence of oxidative stress in bladder UC patients. The levels of all the biochemical parameters were correlated with the grade and severity of the disease. There were significant differences between the patients with low grade tumors and muscle invasive tumors for all parameters (P < 0.001); except AOA (P < 0.279). CONCLUSIONS: The observed redox imbalance in UC of bladder in correlation with the grade and stage, as a consequence of decreased levels of antioxidant vitamins, enzymes, and AOA, along with increased MDA levels in circulation, may be important factors in tumor development and growth. Our results suggest that with advancing stage of bladder UC, the levels of oxidative stress increase, while levels of antioxidant molecules decrease. These findings suggest possible use of antioxidant supplementation as prophylactic agents for prevention and treatment of bladder cancer.

Oxidant-antioxidant status in Indian patients with carcinoma of posterior one-third of tongue.

OBJECTIVE: Cancer of posterior one-third of tongue is seen in 0.43% of total world population. Worldwide, cancer of tongue constitutes 5% of the total cancer incidence. Squamous cell cancer of head and neck is the most common cancer encountered in India. Oxidative stress is potentially harmful to cells and ROS are involved in multistage carcinogenesis, in initiation and promotion. Moreover, the extent of ROS-induced oxidative damage can be exacerbated by decreased efficiency of antioxidant defense mechanisms. The aim of this study was to assess the alterations in the circulating lipid peroxide, antioxidant components and the activities of defense enzymes in patients with cancer of posterior one-third of tongue, with respect to healthy controls in the Indian population. METHODS: 60 patients with newly diagnosed, histologically proven cases of locally advanced squamous cell carcinoma (Stage III-IVa) of posterior one-third of tongue were recruited into the study. 60 healthy controls, without history or laboratory evidence of malignancy and inflammatory disorder, were also included in the study, after obtaining informed consent. Single blood samples were taken from patients, before start of therapy and controls. Lipid peroxides, conjugated dienes, reduced glutathione (GSH), vitamin C and E were estimated using standard methods. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) were assayed using commercially available kits from Randox, UK. RESULTS: The pre-treatment levels of plasma lipid peroxide and conjugated dienes were significantly elevated in patients with carcinoma of posterior one-third of tongue, as compared with controls (p=0.001). Significantly lowered levels of GSH, GPx, SOD and antioxidant vitamins were observed in cancer patients, when compared to control subjects (p=0.001). Pearson's correlation analysis showed a highly statistitically significant negative correlation between pro-oxidant and anti-oxidant levels in patients. CONCLUSION: Increased levels of oxidative stress markers and decreased levels of antioxidants in carcinoma of posterior one-third of tongue suggest that oxidative stress markers play a significant role in the pathophysiology of tongue cancer. These findings may suggest possible use of antioxidant supplementation as prophylactic agents for prevention and treatment of tongue cancer. A larger patient cohort for therapeutic response after treatment with a longer follow-up period studies might yield more significant data on their probable use as predictors of chemoradiosensitivity of cancer of tongue.