MicroRNA biomarkers in leprosy: insights from the Northern Brazilian Amazon population and their implications in disease immune-physiopathology.

Leprosy, or Hansen's Disease, is a chronic infectious disease caused by Mycobacterium leprae that affects millions of people worldwide. Despite persistent efforts to combat it leprosy remains a significant public health concern particularly in developing countries. The underlying pathophysiology of the disease is not yet fully understood hindering the development of effective treatment strategies. However, recent studies have shed light on the potential role of microRNAs (miRNAs), small non-coding RNA molecules that can regulate gene expression, as promising biomarkers in various disease, including leprosy. This study aimed to validate a set of nine circulating miRNAs to propose new biomarkers for early diagnosis of the disease. Hsa-miR-16-5p, hsa-miR-106b-5p, hsa-miR-1291, hsa-miR-144-5p, and hsa-miR-20a-5p showed significant differential expression between non-leprosy group (non-LP) and leprosy group (LP), accurately discriminating between them (AUC > 0.75). In addition, our study revealed gender-based differences in miRNA expression in LP. Notably, hsa-miR-1291 showed higher expression in male LP, suggesting its potential as a male-specific biomarker. Similarly, hsa-miR-16-5p and hsa-miR-20a-5p displayed elevated expression in female LP, indicating their potential as female-specific biomarkers. Additionally, several studied miRNAs are involved in the dysregulation of apoptosis, autophagy, mitophagy, cell cycle, and immune system in leprosy. In conclusion, the validation of miRNA expression highlights several miRNAs as potential biomarkers for early diagnosis and provides new insights into the pathogenesis of the disease.

MicroRNAs correlate with bacillary index and genes associated to cell death processes in leprosy.

Mycobacterium leprae infects skin and peripheral nerves causing a broad of clinical forms. MicroRNAs (miRNAs) control immune mechanisms such as apoptosis, autophagy as well as to target genes leading to abnormal proliferation, metastasis, and invasion of cells. Herein we evaluated miRNAs expression for leprosy phenotypes in biopsies obtained from patients with and without reactions. We also correlated those miRNAs with both, bacillary index (BI) and genes involved in the micobacteria elimination process. Our results show a significant increase in the miR-125a-3p expression in paucibacillary (PB) patients vs multibacillary (MB) subjects (p = 0.007) and vs reversal reactions (RR) (p = 0.005), respectively. Likewise, there was a higher expression of miR-125a-3p in patients with erythema nodosum leprosum (ENL) vs MB without reactions (p = 0.002). Furthermore, there was a positive correlation between miR-125a-3p, miR-146b-5p and miR-132-5p expression and BI in patients with RR and ENL. These miRNAS were also correlated with genes such as ATG12 (miR-125a-3p), TNFRSF10A (miR-146b-5p), PARK2, CFLAR and STX7 (miR-132-5p). All together we underpin a role for these miRNAs in leprosy pathogenesis, implicating mechanisms such as apoptosis and autophagy in skin. The miR-125a-3p might have a distinct role associated with PB phenotype and ENL in MB patients.

Microarray data analysis of antileukemic action of Cinnamoylated benzaldehyde LQB-461 in Jurkat cell line.

BACKGROUND: Leukemias stand out for being the main type of childhood cancer in the world. Current treatments have strong side effects for patients, and there is still a high rate of development of resistance to multidrug therapy. Previously, our research group developed a structure-activity study with novel synthetic molecules analogous to LQB-278, described as an essential molecule with in vitro antileukemic action. Among these analogs, LQB-461 stood out, presenting more significant antileukemic action compared to its derivative LQB-278, with cytostatic and cytotoxicity effect by apoptosis, inducing caspase-3, and increased sub-G1 phase on cell cycle analysis. METHODS AND RESULTS: Deepening the study of the mechanism of action of LQB-461 in Jurkat cells in vitro, a microarray assay was carried out, which confirmed the importance of the apoptosis pathway in the LQB-461 activity. Through real-time PCR, we validated an increased expression of CDKN1A and BAX genes, essential mediators of the apoptosis intrinsic pathway. Through the extrinsic apoptosis pathway, we found an increased expression of the Fas receptor by flow cytometry, showing the presence of a more sensitive population and another more resistant to death. Considering the importance of autophagy in cellular resistance, it was demonstrated by western blotting that LQB-461 decreased LC-3 protein expression, an autophagic marker. CONCLUSIONS: These results suggest that this synthetic molecule LQB-461 induces cell death by apoptosis in Jurkat cells through intrinsic and extrinsic pathways and inhibits autophagy, overcoming some mechanisms of cell resistance related to this process, which differentiates LQB-461 of other drugs used for the leukemia treatment.

Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) contributes to more than 80% of all primary cancers globally and ranks fourth in cancer-related deaths, due to the lack of an effective, definite therapeutic drug. Coleus vettiveroides (CV) has been used in Indian traditional medicine to treat diabetes, liver ailments, skin diseases, leukoderma, and leprosy. This study investigates the anticancer effect of CV ethanolic root extract in HepG2 cells. HepG2 cells were treated with CV extract, and its cytotoxicity was analyzed by MTT assay. AO/EB staining, propidium iodide staining, DCFH-DA assay, phalloidine staining, flow cytometry, and qPCR studies were performed for ROS expression, apoptosis and cell cycle analysis. The phytochemical analysis confirmed the presence of quercetin and galangin in CV root extract. The results showed that CV inhibited the proliferation of HepG2 cells, with altered cellular and nuclear morphology. CV was also found to increase intracellular ROS levels and oxidative stress markers in HepG2 cells. CV significantly altered the actin microfilament distribution in HepG2 cells and caused cell cycle arrest at the sub G0 -G1 phase. CV also induced mitochondria-mediated apoptosis, as evidenced by increased expression of p53, Bax, cytochrome C, Apaf-1, PARP, caspase-3 and caspase-9, and downregulated Bcl-2 expression. Therefore, CV exerts its anticancer effect by inducing mitochondrial dysfunction, oxidative stress, cytoskeletal disorganization, cell cycle arrest, and mitochondria-mediated apoptosis, and it could be a potent therapeutic option for HCC.

Biology and physiology of Hanseniaspora vineae: metabolic diversity and increase flavour complexity for food fermentation.

Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.

Different cell death mechanisms are involved in leprosy pathogenesis.

Leprosy is a chronic granulomatous disease that remains a serious public health problem in developing countries. According to the Madrid classification, leprosy presents in four clinical forms: two immunologically unstable forms (indeterminate and borderline) and two stable polar forms (tuberculoid and lepromatous). In leprosy, the relationship of cell death to clinical disease outcome remains unclear. Therefore, we investigated the extent of autophagy and different cell death mechanisms-such as apoptosis, necroptosis, and pyroptosis-in cutaneous lesions of patients with leprosy, as well as the role of these mechanisms in clinical disease progression. This cross-sectional analytical study included 30 patients with a confirmed diagnosis of leprosy, with 10 patients in each of the following groups: lepromatous (LL), tuberculoid (TT), and indeterminate (II) leprosy groups. For histopathological analysis, skin samples were subjected to haematoxylin-eosin staining and immunostaining for apoptotic and necroptotic markers. The results indicated that FasL expression was much higher in the LL form than in the TT and II forms. Similar results (higher expression in the LL form than in the TT and II forms) were observed for caspase 8, RIP1, and RIP3 expressions. MLKL, BAX, and caspase 3 expression levels were highest in the LL form, especially in globular foamy macrophages. Beclin-1 expression was highest in the TT form but was low in LL and II forms. Caspase 1 expression was highest in the LL form, followed by that in the TT and II forms. In conclusion, our study elucidates the role of different cell death mechanisms in the pathophysiology of various forms of leprosy and suggests measures that may be used to control the host response to infection and disease progression.

Hansenia weberbaueriana (Fedde ex H.Wolff) Pimenov & Kljuykov Extract Suppresses Proliferation of HepG2 Cells via the PTEN-PI3K-AKT Pathway Uncovered by Integrating Network Pharmacology and Iin Vitro Experiments.

Previous studies have shown that Hansenia weberbaueriana (Fedde ex H.Wolff) Pimenov & Kljuykov extracts (HWEs) have antitumor activity, but their mechanism in vitro is still unclear. In this study, we first combined network pharmacology with experimental evaluation and applied a comprehensive strategy to explore and prove the therapeutic potential and potential mechanism of HWE. The mRNA expression profiles of PTEN, PIK3A, and AKT1 are from the Cancer Cell Line Encyclopedia (CCLE) of the Broad Institute. Our results showed that HWE has a good inhibition on HepG2 cells, and a slight inhibition on other cells. The results of the CCLE database showed that PTEN/PIK3A/AKT1 mRNA expression was up-regulated in HepG2 cells. Through further study, it was found that HWE increased the release of LDH, induced early and late apoptosis, and increased ROS levels in HepG2 cells. Western blot showed that HWE regulates the expression of mitochondrial apoptosis-related proteins. Meanwhile, the expression of PTEN was increased, and the expression of phosphorylated PI3K and Akt was down-regulated after HWE treatment. Our results show that HWE promotes HepG2 cell apoptosis via the PTEN-PI3K-Akt signaling pathway. This study is the first to report the potential role of HWE in the treatment of liver cancer.

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Clinical and pathological characterization of oral mucosal 'lichen planus-like lesions' in patients with pemphigus vulgaris: An observational study.

BACKGROUND: Lichen planus-like lesions on oral mucosa occasionally occur in Indian patients with pemphigus vulgaris. Its significance, both clinical and pathological, is yet to be elucidated. AIMS AND OBJECTIVES: To study the clinical and pathological characteristics of clinically apparent oral mucosal lichen planus-like lesions in pemphigus patients and to assess their relation with pemphigus disease activity. MATERIALS AND METHODS: A total of 32 patients with pemphigus vulgaris who had oral lichen planus-like lesions were included and classified as 'cases,' and eight diagnosed cases of pemphigus vulgaris without lichenoid 'hue' were included as controls. The biopsy specimens were subjected to routine histopathologic examination, immunohistochemistry with FasL, and caspase-3 and direct immunofluorescence. RESULTS: On histopathologic examination, the diagnosis of pemphigus vulgaris, lichen planus, 'overlap' and 'nonspecific' were rendered in 19 (59.4%), 4 (12.5%), 5 (15.6%) and 4 (12.5%) cases, respectively. On immunohistochemistry, FasL was positive in epithelial cells in 16 (50%) cases and 4 (12.5%) controls (P = 0.066). Caspase-3 stained positively in 18 (56.2%) cases and 20 (62.5%) controls (P = 0.77). Direct immunofluorescence was positive in 77.8% (21/27) of the cases. LIMITATIONS: Relatively small number of controls is the limitation of this study. CONCLUSION: Lichen planus-like lesions in pemphigus should not be labeled as inactive disease or postinflammatory hyperpigmentation. Apoptosis followed by pigment incontinence seems to explain such lesions with 'lichen planus-like appearance' in oral pemphigus vulgaris. Active pemphigus smoulders in a majority of these lesions.

microRNAs in Mycobacterial Infection: Modulation of Host Immune Response and Apoptotic Pathways.

Our current knowledge of mycobacterial infections in humans has progressively increased over the past few decades. The infection of Mycobacterium tuberculosis causes tuberculosis (TB) disease, which has reasoned for excessive morbidity and mortality worldwide, and has become a foremost issue of health problem globally. Mycobacterium leprae, another member of the family Mycobacteriaceae, is responsible for causing a chronic disease known as leprosy that mainly affects mucosa of the upper respiratory tract, skin, peripheral nerves, and eyes. Ample amount of existing data suggests that pathogenic mycobacteria have skilled in utilizing different mechanisms to escape or offset the host immune responses. They hijack the machinery of immune cells through the modulation of microRNAs (miRs), which regulate gene expression and immune responses of the host. Evidence shows that miRs have now gained considerable attention in the research, owing to their involvement in a broad range of inflammatory processes that are further implicated in the pathogenesis of several diseases. However, the knowledge of functions of miRs during mycobacterial infections remains limited. This review summarises recent findings of differential expression of miRs, which are used to good advantage by mycobacteria in offsetting host immune responses generated against them.

Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and reactive oxygen species generation in A549 and NCI-H1299 non-small cell lung cancer cells.

BACKGROUND: Calotropis gigantea (CG) is a tall and waxy flower that is used as a traditional remedy for fever, indigestion, rheumatism, leprosy, and leukoderma. However, the precise mechanisms of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells. In this study, we investigated whether CG extract exerted an apoptotic effect in A549 and NCI-H1299 NSCLC cells. METHODS: The ethanol extract of CG was prepared, and its apoptotic effects on A549 and NCI-H1299 NSCLC cells were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining, cell cycle analysis, real-time polymerase chain reaction (RT-PCR), western blotting, JC-1 staining, and ROS detection assay. RESULTS: The CG extract induced apoptosis through the stimulation of intrinsic and extrinsic signaling pathways in A549 and NCI-H1299 lung cancer cells. Cell cycle arrest was induced by the CG extract in both cell lines. Reactive oxygen species (ROS), which can induce cell death, were also generated in the CG-treated A549 and NCI-H1299 cells. CONCLUSIONS: These data confirmed that CG caused apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cells. Thus, CG can be suggested as a potential agent for lung cancer therapy.

In Vitro Anti-proliferative Activity and Mechanism of Action of Anemone nemorosa.

Anemone nemorosa is part of the Ranunculaceae genus Anemone (order Ranunculales) which comprises more than 150 species. Various parts of the plant have been used for the treatment of numerous medical conditions such as headaches, tertian agues, rheumatic gout, leprosy, lethargy, eye inflammation as well as malignant and corroding ulcers. The Anemone plants have been found to contain various medicinal compounds with anti-cancer, immunomodulatory, anti-inflammatory, anti-oxidant and anti-microbial activities. To date there has been no reported evidence of its use in the treatment of cancer. However, due to the reported abundance of saponins which usually exert anti-cancer activity via cell cycle arrest and the induction of apoptosis, we investigated the mode of cell death induced by an aqueous A. nemorosa extract by using HeLa cervical cancer cells. Cisplatin was used as a positive control. With a 50% inhibitory concentration (IC50) of 20.33 ± 2.480 µg/mL, treatment with A. nemorosa yielded a delay in the early mitosis phase of the cell cycle. Apoptosis was confirmed through fluorescent staining with annexin V-FITC. Apoptosis was more evident with A. nemorosa treatment compared to the positive control after 24 and 48 h. Tetramethylrhodamine ethyl ester staining showed a decrease in mitochondrial membrane potential at 24 and 48 h. The results obtained imply that A. nemorosa may have potential anti-proliferative properties.

Live Mycobacterium leprae inhibits autophagy and apoptosis of infected macrophages and prevents engulfment of host cell by phagocytes.

Previous studies demonstrated that live Mycobacterium leprae (M. leprae) infection promoted macrophage differentiation toward the M2 type, with elevated interleukin (IL)-10 production. The underlying mechanism is not entirely clear. In this study, we treated macrophages with primary M. leprae strains isolated from both lepromatous leprosy (L-lep) and tuberculoid leprosy (T-lep) patients. We found that infection by live M. leprae, regardless of the primary strain, resulted in M2 skewing in the infected macrophage. This skewing was associated with downregulated IRGM expression, a core organizer protein in the autophagy assembly and reduced autophagosome formation, and with lower annexin V staining and lower caspase 3 and caspase 9 activity. Moreover, live M. leprae-infected macrophages prevented efficient phagocytosis by uninfected bystander macrophages. As a result, the phagocytes secreted less pro-inflammatory cytokines, and preferentially primed anti-inflammatory T cell responses. Together, these results suggested that live M. leprae could employ a strain-independent mechanism to suppress inflammation, possibly involving the inhibition of autophagy and apoptosis in the infected macrophages.

Protective effects of glutamine on human melanocyte oxidative stress model.

BACKGROUND: Vitiligo is a disorder caused by the loss of the melanocyte activity on melanin pigment generation. Studies show that oxidative-stress induced apoptosis in melanocytes is closely related to the pathogenesis of vitiligo. Glutamine is a well known antioxidant with anti-apoptotic effects, and is used in a variety of diseases. However, it is unclear whether glutamine has an antioxidant or anti-apoptotic effect on melanocytes. AIMS: The aim of this study was to investigate the protective effects of glutamine on a human melanocyte oxidative stress model. METHODS: The oxidative stress model was established on human melanocytes using hydrogen peroxide. The morphology and viability of melanocytes, levels of oxidants [reactive oxygen species and malondialdehyde], levels of antioxidants [superoxide dismutase and glutathione-S-transferase], and apoptosis-related indicators (caspase-3, bax and bcl-2) were examined after glutamine exposure at various concentrations. Expressions of nuclear factor-E2-related factor 2, heme oxygenase-1, and heat shock protein 70 were detected using western blot technique after glutamine exposure at various concentrations. RESULTS: Our results demonstrate that pre-treatment and post-treatment with glutamine promoted melanocyte viability, increased levels of superoxide dismutase, glutathione-S-transferase and bcl-2, decreased levels of reactive oxygen species, malondialdehyde, bax and caspase-3, and enhanced nuclear factor-E2-related factor 2, heme oxygenase-1, and heat shock protein 70 expression in a dose dependent manner. The effect of pre-treatment was more significant than post-treatment, at the same concentration. LIMITATIONS: The mechanisms of glutamine activated nuclear factor-E2-related factor 2 antioxidant responsive element signaling pathway need further investigation. CONCLUSIONS: Glutamine enhances the antioxidant and anti-apoptotic capabilities of melanocytes and protects them against oxidative stress.

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology.

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1, and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial-mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1, and aquaporin-1 (AQP1) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain.

A new clerodane furano diterpene glycoside from Tinospora cordifolia triggers autophagy and apoptosis in HCT-116 colon cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia is a miraculous ayurvedic herb used in the treatment of innumerable diseases such as diabetes, gonorrhea, secondary syphilis, anaemia, rheumatoid arthritis, dermatological diseases, cancer, gout, jaundice, asthma, leprosy, in the treatment of bone fractures, liver & intestinal disorders, purifies the blood, gives new life to the whole body; (rejuvenating herb) and many more. Recent studies have revealed the anticancer potential of this plant but not much work has been done on the anticancer chemical constituents actually responsible for its amazing anticancer effects. This prompted us to investigate this plant further for new potent anticancer molecules. AIM OF THE STUDY: The present study was designed to isolate and identify new promising anticancer candidates from the aqueous alcoholic extract of T. cordifolia using bioassay-guided fractionation. MATERIALS AND METHODS: The structures of the isolated compounds were determined on the basis of spectroscopic data interpretation and that of new potent anticancer molecule, TC-2 was confirmed by a single-crystal X-ray crystallographic analysis of its corresponding acetate. The in vitro anti-cancer activity of TC-2 was evaluated by SRB assay and the autophagic activity was investigated by immunofluorescence microscopy. Annexin-V FITC and PI dual staining was applied for the detection of apoptosis. The studies on Mitochondrial Membrane potential and ROS (Reactive oxygen species) production were also done. RESULTS: Bioassay guided fractionation and purification of the aqueous alcoholic stem extract of Tinospora cordifolia led to the isolation of a new clerodane furano diterpene glycoside (TC-2) along with five known compounds i.e. cordifolioside A (ß-D-Glucopyranoside,4-(3-hydroxy-1-propenyl)- 2,6-dimethoxyphenyl 3-O-D-apio-ß-D-furanosyl) (TC-1), ß-Sitosterol(TC-3), 2ß,3ß:15,16-Diepoxy- 4α, 6ß-dihydroxy-13(16),14-clerodadiene-17,12:18,1-diolide (TC-4), ecdysterone(TC-5) and tinosporoside(TC-6). TC-2 emerged as a potential candidate for the treatment of colon cancer. CONCLUSION: The overall study on the bioassay guided isolation of T.cordifolia identified and isolated a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. We have reported a promising future candidate for treating colon cancer.

Anti-cancer effect of clofazimine as a single agent and in combination with cisplatin on U266 multiple myeloma cell line.

Multiple Myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effect on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancer as well as melanoma, neuroblastoma, and leukemia. The objective of this study was to evaluate the anticancer effect and mechanism of CLF on U266 MM cell line. CLF (10µM, 24h) treatment resulted up to 72% growth suppression on a panel of hematological cell lines. Dose-response study conducted on U266 MM cell line revealed an IC50 value of 9.8±0.7µM. CLF also showed a synergistic inhibition effect in combination with cisplatin. In mechanistic assays, CLF treatment caused mitochondrial membrane depolarization, change in cell membrane asymmetry and increase in caspase-3 activity; indicating to an intrinsic apoptosis mechanism. This study provides new evidence for the anticancer effect of CLF on U266 cell line. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for MM treatment.

Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries. Unfortunately, the overall 5-year survival rate is still less than 5%. The most frequent oncogenic mutations in PDAC are loss-of function mutations in p53 and gain-of-function mutations in KRAS. Here we show that clofazimine (Lamprene), a drug already used in the clinic for autoimmune diseases and leprosy, is able to efficiently kill in vitro five different PDAC cell lines harboring p53 mutations. We provide evidence that clofazimine induces apoptosis in PDAC cells with an EC50 in the µM range via its specific inhibitory action on the potassium channel Kv1.3. Intraperitoneal injection of clofazimine resulted in its accumulation in the pancreas of mice 8 hours after administration. Using an orthotopic PDAC xenotransplantation model in SCID beige mouse, we show that clofazimine significantly and strongly reduced the primary tumor weight. Thus, our work identifies clofazimine as a promising therapeutic agent against PDAC and further highlights ion channels as possible oncological targets.

Evaluation of apoptosis in skin biopsies of patients of borderline leprosy and lepra type 1 reaction.

BACKGROUND: The role of apoptosis is not clear in leprosy and lepra reactions. OBJECTIVES: To evaluate frequency of apoptosis in skin lesions of borderline leprosy and Type 1 lepra reaction. METHODS: Sixty patients with borderline leprosy (30 with clinically diagnosed Type 1 reaction (T1R) (Group I) and 30 without clinical evidence of reaction (Group II)) were analyzed in this prospective study. Apoptosis was detected by two different methods for comparison, that is, histopathologic examination (HPE) and deoxyribonucleic acid (DNA) fragmentation and electrophoresis. Quantification of apoptotic bodies/10 high power fields (HPF) was also done. RESULTS: Out of 30 cases, apoptosis was detected in 29 cases in Group I and 24 cases in Group II by HPE (P = 0.103), whereas, with the use of DNA electrophoresis it was detected in 24 cases in Group I and 18 cases in Group II (P = 0.091). On quantitative estimation it was found that number of apoptotic bodies are higher in Group I in comparison to Group II (2.77 vs 1.99), which is statistically significant. CONCLUSIONS: There was moderate agreement (κ = 0.47) between the two methods of apoptosis detection. Apoptosis was seen more in patients with T1R both qualitatively (statistically nonsignificant) and quantitatively (statistically significant). Clinical significance of this novel finding is that apoptosis can be used as one of the variables for diagnosis of T1R to increase detection rate.

Apoptotic activity and Treg cells in tissue lesions of patients with leprosy.

In order to understand the apoptotic response and the participation of Treg cells in the spectral clinical evolution of leprosy, this study evaluated the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of leprosy patients with the polar forms of the disease. Forty-nine patients with a confirmed diagnosis of the disease were selected, including 27 with the TT form and 22 with the LL form. Quantitative analysis of caspase-3 immunostaining showed a higher expression of this mediator in the LL form (3.409 ± 0.6517 cells/mm(2); p = 0.0001). Immunostaining for the transcription factor FoxP3 was higher in the LL form (3.891 ± 0.9294 cells/mm(2); p = 0.0001). A moderate correlation between the two markers was observed in the TT form (r = 0.5214; p = 0.005). It can be concluded that Treg cells and apoptosis play an effective role for the host defense response, inducing mechanisms involved in the activation of cascades that interfere with the control of the immune response and cell homeostasis.