Cervicodorsal spine tuberculosis-- surgical approach.

Background: Tuberculosis (TB) of CT junction is uncommon (5 % of all spinal TB), and difficult to approach surgically in view of its deep location with sternum in front and scapula in the back. We present 7 consecutively treated cases of cervico-thoraccic TB for outcome of treatment and discuss rationale of choosing surgical approach. Methods: Present study includes 7 freshly diagnosed cases of CT junction TB. Plain radiographs, sagittal reconstruction of CT spine that included sternum on CT/MRI was performed in all cases. Disc space below the distal healthy vertebrae was identified and a line parallel to disc space was drawn. If this line passes above suprasternal notch, it was inferred that this VB can be accessed by anterior cervical approach. If disease focus was at or below suprasternal notch level, manubriotomy/sternotomy was added for better visualization of the lesion. Results: All seven cases were female, with mean age of 20 years (9-45 years). The vertebral lesion involved 2VB (n = 3), 3VB (n = 2) and >3 VB (n = 2). The average Cervico-thoracic kyphosis was 15° (range 10-25°). All 7 cases were operated for anterior decompression, kyphotic deformity correction and instrumented stabilization. Anterior cervical approach and manubriotomy/sternotomy approach was performed in three cases each. In two pan-vertebral cases we performed 360° procedure. Six cases have shown first sign of neural recovery within 3 weeks of surgery and almost complete neural recovery at 3 months follow-up while one case showed partial recovery. ATT was stopped after 12 months once healed stage was demonstrated on contrast MRI in all. Conclusions: CT junction TB usually presents with severe kyphotic deformity/neural deficit. These cases require anterior decompression/corpectomy, deformity correction, gap grafting and instrumented stabilization with anterior cervical plate. Lesion with pan-vertebral disease is stabilized 360°. These lesions can be decompressed by lower anterior cervical approach with/without manubriotomy. The Karikari method was useful in deciding the need for manubriotomy to decompress the lesion.

Incidence and Patterns of Drug Resistance in Patients with Spinal Tuberculosis: a Prospective, Single-Center Study from a Tuberculosis-Endemic Country.

Background: There is paucity of data on incidence and pattern of drug resistance in spinal TB. This prospective observational study was conducted to document the incidence and drug-resistance pattern among primary and presumptive resistant cases. Methods: 59 consecutive cases diagnosed clinico-radiologically (imaging) were grouped into Group A (n = 51, primary cases) and Group B (n = 8, presumptive resistant cases) based on pre-defined criteria (INDEX-TB guidelines). Tissue samples obtained percutaneously (37.29%, 22/59) and on surgery (62.71%, 37/59) were subjected to genotypic DST (CBNAAT, LPA) and phenotypic DST (BACTEC MGIT 960 culture and sensitivity using fixed critical concentration of drugs). Results: Etiological diagnosis was ascertained in all. 13/51 (25.49%) in Group A, while 3/8 (37.5%) in Group B and 16/59 (27.12%) overall demonstrated drug resistance. 12/16 (75%) had no prior history of ATT intake. 4 demonstrated INH (Isoniazid) mono-resistance. 12 polydrug resistance demonstrated: 5MDR, 3pre-XDR, while RIF + FQ (fluoroquinolones), FQ + Lz (linezolid), only SLID (second-line injectable drugs), and only FQ resistance observed in 1 case each. Isolated RIF (Rifampicin) resistance and XDR pattern were not observed. Overall frequency of RIF resistance was 16.4% (9/55) and INH was 25% (12/48) with low-(n-2) and high-level INH resistance (n-10). Among second-line drugs, FQ resistance was more than SLID resistance and within FQ, levofloxacin resistance was more frequent than moxifloxacin. MGIT demonstrated positive growth in 16/59 samples, out of which 1 sample was positive for nontuberculous mycobacteria (M. chelonae) but on genotypic testing demonstrated MTB resistant to RIF and FQ. Conclusion: This is the first report on incidence and drug-resistant pattern in culture-positive/negative cases. High (25.49%) primary drug resistance is worrisome. This being the first study in  spinal TB cases which document prevalent drug-resistant pattern as evaluated for consecutive culture-positive/negative cases. The tissue obtained must be submitted for AFB culture and molecular tests to ascertain drug resistance in culture-positive/negative cases. However, in the presence of insufficient tissue sample histology and CBNAAT can ascertain etiological diagnosis in 100% cases. INH resistance is more than RIF with isolated RIF resistance unreported.

Drug Resistance in Osteoarticular Tuberculosis: A Study From an Endemic Zone.

BACKGROUND: The present study was undertaken to determine the incidence of drug resistance against anti-tubercular drugs among patients from an endemic zone.  Methodology: Forty consecutive clinico-radiologically diagnosed patients of osteoarticular tuberculosis (29: spine, 11: extraspinal) were enrolled. Pus from needle aspiration was taken in 31 cases, tissue following spinal decompression in seven, synovial in one, and sinus edge biopsy in one. The pus/tissue was subjected to acid-fast bacilli (AFB) staining and liquid culture, sensitivity to 13 anti-tubercular drugs (Isoniazid (INH), rifampicin (RIF), kanamycin (KAN), amikacin (AMK,) capreomycin (CAP), ethionamide (ETH), levofloxacin (LEV), moxifloxacin (MOX), linezolid (LNZ), para-amino-salicylic acid (PAS), bedaquiline (BDQ), delamanid (DLM), and clofazimine (CFO)) were checked, and histopathological/cytopathological examination and molecular tests were performed.   Results: The mean age of patients was 29.07(9-65) years; 21 were female and 19 were male. The diagnostic accuracy for tuberculosis was 20% by AFB smear, 65% by liquid culture, 82.5% by histopathology, and 90% by cartridge-based nucleic acid amplification testing (CBNAAT). All culture-positive isolates were identified as Mycobacterium tuberculosis with no non-tubercular Mycobacterium. The drug resistance detected on CBNAAT was 11.1%, line probe assay (LPA) first line was 15.4%, LPA second line was 4%, and liquid drug susceptibility testing (DST) 11.5%. We detected 15.4% INH resistance, 11.1% RIF, 7.6% LEV, 3.8% MOX and PAS. No resistance was detected against second-line injectable drugs (SLID), ETH, LNZ, BDQ, DLM, and CFO.    Conclusions: No single laboratory modality can ascertain the diagnosis in all cases; hence, samples should be sent for all tests in tandem. In the presence of insufficient samples, tissue may be subjected to CBNAAT and histopathology to arrive at tissue diagnosis. In this subset, overall drug resistance incidence was 12.5% (5/40) with one patient each of isolated INH and RIF resistance, one of multidrug-resistance (MDR), and two of pre-extensively drug-resistant (pre-XDR). Primary drug resistance came out to be 11.1% (4/36) with one patient each of isolated INH and RIF resistance, one of MDR, and one Pre-XDR.

Stage-Specific Effect of Inositol Hexaphosphate on Cancer Stem Cell Pool during Growth and Progression of Prostate Tumorigenesis in TRAMP Model.

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8-15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55-70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage.

Clinical outcome and computer tomography based tunnel placement evaluation following arthroscopic anteromedial portal anterior cruciate ligament reconstruction in non-athletic population.

BACKGROUND: The morphology of distal femur and proximal tibia varies between different ethnicities, and it can affect the tunnel dimensions and positions while doing ACL reconstruction which may affect the clinical outcome. There is limited data on the clinical outcome and CT based tunnel placement evaluation in femur and tibia of Indian nonathletic population. METHODS: Thirty non-athletic patients with mean age of 25.50±6.9 years and ACL rupture who underwent single bundle hamstring autograft arthroscopic ACL reconstruction by anteromedial portal were included in the study. Their preoperative IKDC Score, Lysholm-Tegner score, Tegner activity level were calculated and knee stability was assessed clinically using anterior drawer test, Lachman test and pivot shift test. The CT scan of the operated knee was done once the complete extension of the knee was achieved. Using the multimodality workstation available at the department of radio-diagnosis the tunnel parameters of femoral and tibial tunnel was calculated. After 6 months the patients were reassessed for clinical and radiological outcome. The postoperative outcome was compared with preoperative outcome. RESULTS: There was a significant difference in preoperative and postoperative score, the difference in IKDC score was 15.08 points, improvement of 14.65 points was seen in Lysholm-Tegner score and there was marked improvement in Tegner activity level. Tests for knee stability were normal in >90% of patients postoperatively. The CT evaluation showed that the femoral tunnels were positioned at 28.45%±3.69% (20.16%-38.35%) along the deep-shallow axis and 25.81%±3.819% (20.69%-37.35%), the mean tunnel obliquity compared to the femoral shaft axis were 47.34°±5.427° (37.68°-58.16°) in the coronal plane and 47.93°±7.023° (35.11°-63.95°), the mean tunnel length was 3.38 cm±0.331 cm (2.79 cm-4.18 cm). The tibial tunnel were positioned at 45.63%±5.832% (32.23%-58.23%) along the anterior-posterior axis and 47.70%±2.26% (42.40%-51.96%) along the medio-lateral axis. The tibial tunnel length was found to be 3.89 cm±0.519 cm (3.05 cm-5.06 cm). CONCLUSION: This study helps to ascertain that the ACL reconstruction via anteromedial portal technique using femoral offset zig followed by postoperative home-based rehabilitation technique gives favorable clinical outcomes in Indian non-athletic patients. All patients had improvement in stability of knee after the surgery. The position of femoral tunnels was anatomical but in comparison to Caucasian patients its placement was deeper and higher. Hence, we conclude that the anteromedial portal technique of ACL reconstruction provides favorable clinical outcome and adequate anatomical tunnel placement in Indian non athletic patients.

Extended Curettage and Fibular Grafting in Enchondroma of the Acromion.

We report the case of a six-year-old male child with progressive pain and swelling of the right shoulder for six months. On examination, there was a 7x7 cm globular, tender swelling with firm consistency over the posterolateral corner of the right shoulder. Radiographs showed an expansile lytic lesion in the acromion process of the scapula. Biopsy showed lobules of hypocellular cartilage separated by fibroconnective stroma suggestive of an enchondroma. Extended curettage and fibular bone grafting of the lesion was done. At one-year follow-up, the patient was symptom-free and had full, painless shoulder range of motion. To the best of our knowledge, there is no published record of an enchondroma of the acromion.

Outcomes of Core Decompression with or without Nonvascularized Fibular Grafting in Avascular Necrosis of Femoral Head: Short Term Followup study.

BACKGROUND: Avascular necrosis (AVN) of the femoral head usually occurs in the third to fifth decade. The treatment options depend on the stage of disease varying from nonoperative treatment to surgical procedure including core decompression (CD) with or without nonvascularized or vascularized fibular graft, muscle pedicle bone grafting, osteotomies, and arthroplasty. Finite life of the total hip arthroplasty (THA) prosthesis limits its use in young adults or in middle aged. In this study, we envisage to evaluate the clinicoradiological outcomes of CD and nonvascularized fibular grafting in early stages of AVN femoral head. MATERIALS AND METHODS: Our study is longitudinal observational study including 76 hips (46 patients) in the age group of 18-48 years (mean 30.07 years). Ficat and Arlet staging system was used and only early stages, that is, Stage 1 (n = 36 hips) and Stage 2 (n = 40 hips) were included in the study. The cases with traumatic AVN were excluded. All patients in Stage 1 underwent CD (Group 1) and those in Stage 2 underwent CD and fibular grafting (Group 2). Preoperative Harris Hip Score (HHS), visual analog score (VAS), plain radiographs, and magnetic resonance imaging (MRI) were compared with serial postoperative HHS, VAS, plain radiographs, and MRI taken at different intervals. RESULTS: Average period of followup was 53.5 months (44-63 months). Radiological progression was not seen in 55 hips out of 76 hips (72.3%), whereas 21 hips (27.6%) demonstrated signs of progression and collapse. Failure of surgery was defined as progression of the disease, which was 25% (n = 9) in Group 1and 30% (n = 12) Group 2. Median values of HHS at the end of the followup in Group 1 was 77 and in Group 2 was 71.5 compared to the preoperative HHS of 48 and 62 in Group 1 and 2, respectively. Median values of VAS at the end of the followup in Stage 1 was 0 and in Stage 2 was 2 compared to the preoperative VAS of 6 and 8 in Group 1 and Group 2, respectively. CONCLUSION: CD with or without fibular grafting is effective in preserving the sphericity of the femoral head and to delay the progression of the AVN of femoral head in the early stages, that is, Stage 1 and Stage 2 and aids in the early revascularization of ischemic femoral head and is a useful modality to negate or delay the requirement of THA.

Outcome of Percutaneous Fixation of Calcaneal Fractures: A Prospective Analysis in an Indian Population.

Displaced intra-articular calcaneal fractures can be difficult to treat. Open surgical techniques are associated with wound complications, whereas nonoperative management leads to arthrosis. In the present study, 23 displaced intra-articular calcaneal fractures in 19 patients were treated with closed reduction and percutaneous Kirschner wire fixation. Sanders and Essex-Lopresti classification systems were used. We studied anatomical (Gissane and Bohler angles and width of calcaneus) and functional (Maryland Foot Score and American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Score) outcomes after 6, 18, and 26 months. Mechanism of injury, fluoroscopy use, time since injury, time delay to surgery, method of reduction, and number of Kirschner wires used were recorded. The mean participant age was 29.5 (17 to 46) years, mean delay to surgery was 7 (2 to 12) days, mean length of surgery was 61 (range 20 to 175) minutes, and mean fluoroscopy time was 115 (range 20 to 254) seconds. All patients were followed for a minimum of 26 months, and the mean duration of follow-up was 32.4 (26 to 36) months. There were 18 (78.26%) joint depression and 5 (21.74%) tongue-type fractures, whereas there were 2 (8.69%) Sanders type II, 13 (56.52%) Sanders type III, and 8 (34.78%) Sanders type IV fractures. The mean Maryland Foot Score and American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Score at 6 months were 86.7 (81 to 92) and 84.2 (75 to 93), whereas at 26 months, the scores were 87.7 (82 to 93) and 85.1 (75 to 94), respectively. No pin site infections, cases of sural nerve dysfunction, or revision/additional surgery was experienced, and 17 (86.6%) patients were able to return to their original occupation at the end of 26 months.

Tuberculosis - 'The great masquerader' presenting as a dumb-bell-shaped intradural extramedullary tumor in a 20-year-old female.

Tuberculosis has been known as the great masquerader for its varied presentations. We present an extraordinary case of a 20-year-old female who presented with paraparesis of two months. MRI showed an intradural, extramedullary dumb-bell-shaped, spinal cord tumor. With a provisional clinicoradiological diagnosis of benign nerve sheath tumor (schwannoma/neurofibroma), laminectomy was done. But after durotomy, frank pus was drained from the site of lesion and the laboratory investigations of the tissue and pus obtained proved it to be tubercular. This is a rare case reported in the literature where tuberculosis is mimicking as a dumb-bell-shaped, spinal cord tumor.

Role of p53 in silibinin-mediated inhibition of ultraviolet B radiation-induced DNA damage, inflammation and skin carcinogenesis.

Non-melanoma skin cancers (NMSC) are a growing problem given that solar ultraviolet B (UVB) radiation exposure is increasing most likely due to depletion of the atmospheric ozone layer and lack of adequate sun protection. Better preventive methods are urgently required to reduce UV-caused photodamage and NMSC incidence. Earlier, we have reported that silibinin treatment activates p53 and reduces photodamage and NMSC, both in vitro and in vivo; but whether silibinin exerts its protective effects primarily through p53 remains unknown. To address this question, we generated p53 heterozygous (p53+/-) and p53 knockout (p53-/-) mice on SKH-1 hairless mouse background, and assessed silibinin efficacy in both short- and long-term UVB exposure experiments. In the chronic UVB-exposed skin tumorigenesis study, compared to p53+/+ mice, p53+/- mice developed skin tumors earlier and had higher tumor number, multiplicity and volume. Silibinin topical treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but silibinin' protective efficacy was significantly compromised in p53+/- mice. Additionally, silibinin treatment failed to inhibit precursor skin cancer lesions in p53-/- mice but improved the survival of the mice. In short-term studies, silibinin application accelerated the removal of UVB-induced DNA damage in p53+/+ mice while its efficacy was partially compromised in p53-/- mice. Interestingly, silibinin treatment also inhibited the UVB-induced inflammatory markers in skin tissue. These results further confirmed that absence of the p53 allele predisposes mice to photodamage and photocarcinogenesis, and established that silibinin mediates its protection against UVB-induced photodamage, inflammation and photocarcinogenesis partly through p53 activation.

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc.

Beneficial effects of the naturally occurring flavonoid silibinin on the prostate cancer microenvironment: role of monocyte chemotactic protein-1 and immune cell recruitment.

Tumor microenvironment plays an essential role in prostate carcinogenesis and offers novel opportunities to prevent and treat prostate cancer (PCA). Here, we investigated the ability of cancer-associated fibroblasts (CAFs) to promote PCA progression, and silibinin efficacy to target this response. We collected conditioned media from CAFs treated with vehicle or silibinin, and labeled as control conditioned media (CCM) or silibinin-treatment conditioned media (SBCM), respectively. Next, we characterized the effect of CCM and SBCM treatment in several PCA cell lines (RWPE-1, WPE-1 NA-22, WPE-1 NB-14 and PC3). Result showed that compared with SBCM, CCM significantly reduces E-cadherin expression and increases invasiveness and clonogenicity in PCA cells. Further molecular studies identified monocyte chemotactic protein-1 (MCP-1) as the key component of CCM that promotes PCA invasiveness, whereas silibinin treatment strongly reduced MCP-1 expression in CAFs by inhibiting the DNA-binding activity of MCP-1 transcriptional regulators-nuclear factor-kappaB and AP-1. In vivo, silibinin feeding (200mg/kg body weight) strongly reduced TRAMPC1 allografts growth (by 68%) in syngeneic C57Bl/6 mice. TRAMPC1 tumor analysis showed that silibinin reduced MCP-1 and CAFs' biomarkers (fibroblast activation protein, α-smooth muscle actin, transforming growth factor beta 2, vimentin etc.) and significantly modulated the recruitment of immune cells in the tumor microenvironment. Similar inhibitory effects of silibinin on MCP-1 and immune cells recruitment were also observed in TRAMP PCA tissues with reported silibinin efficacy. Overall, our data suggest that silibinin can target CAF-mediated invasiveness in PCA by inhibiting MCP-1 secretion. This, in turn, was associated with a reduction in immune cell recruitment in vivo along with a marked reduction in tumor growth.

Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 µg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

A Fast and Accurate Unconstrained Face Detector.

We propose a method to address challenges in unconstrained face detection, such as arbitrary pose variations and occlusions. First, a new image feature called Normalized Pixel Difference (NPD) is proposed. NPD feature is computed as the difference to sum ratio between two pixel values, inspired by the Weber Fraction in experimental psychology. The new feature is scale invariant, bounded, and is able to reconstruct the original image. Second, we propose a deep quadratic tree to learn the optimal subset of NPD features and their combinations, so that complex face manifolds can be partitioned by the learned rules. This way, only a single soft-cascade classifier is needed to handle unconstrained face detection. Furthermore, we show that the NPD features can be efficiently obtained from a look up table, and the detection template can be easily scaled, making the proposed face detector very fast. Experimental results on three public face datasets (FDDB, GENKI, and CMU-MIT) show that the proposed method achieves state-of-the-art performance in detecting unconstrained faces with arbitrary pose variations and occlusions in cluttered scenes.

Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 µmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer.

Nitrogen mustard exposure of murine skin induces DNA damage, oxidative stress and activation of MAPK/Akt-AP1 pathway leading to induction of inflammatory and proteolytic mediators.

Our recent studies in SKH-1 hairless mice have demonstrated that topical exposure to nitrogen mustard (NM), an analog of sulfur mustard (SM), triggers the inflammatory response, microvesication and apoptotic cell death. Here, we sought to identify the mechanism/s involved in these NM-induced injury responses. Results obtained show that NM exposure of SKH-1 hairless mouse skin caused H2A.X and p53 phosphorylation and increased p53 accumulation, indicating DNA damage. In addition, NM also induced the activation of MAPKs/ERK1/2, JNK1/2 and p38 as well as that of Akt together with the activation of transcription factor AP1. Also, NM exposure induced robust expression of pro-inflammatory mediators namely cyclooxygenase 2 and inducible nitric oxide synthase and cytokine tumor necrosis factor alpha, and increased the levels of proteolytic mediator matrix metalloproteinase 9. NM exposure of skin also increased lipid peroxidation, 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation, protein and DNA oxidation indicating an elevated oxidative stress. We also found NM-induced increase in the homologous recombinant repair pathway, suggesting its involvement in the repair of NM-induced DNA damage. Collectively, these results indicate that NM induces oxidative stress, mainly a bi-phasic response in DNA damage and activation of MAPK and Akt pathways, which activate transcription factor AP1 and induce the expression of inflammatory and proteolytic mediators, contributing to the skin injury response by NM. In conclusion, this study for the first time links NM-induced mechanistic changes with our earlier reported murine skin injury lesions with NM, which could be valuable to identify potential therapeutic targets and rescue agents.

Asiatic acid induces endoplasmic reticulum stress and apoptotic death in glioblastoma multiforme cells both in vitro and in vivo.

Glioblastoma multiforme (GBM) is an untreatable malignancy. Existing therapeutic options are insufficient, and adversely affect functional and non-cancerous cells in the brain impairing different functions of the body. Therefore, there is an urgent need for additional preventive and therapeutic non-toxic drugs against GBM. Asiatic acid (AsA; 2,3,23-trihydroxy-12-ursen-28-oic acid, C30 H48 O5 ) is a natural small molecule widely used to treat various neurological disorders, and the present research investigates AsA's efficacy against GBM both in vitro and in vivo. Results showed that AsA treatment (10-100 µM) decreased the human GBM cell (LN18, U87MG, and U118MG) viability, with better efficacy than temozolomide at equimolar doses. Orally administered AsA (30 mg/kg/d) strongly decreased tumor volume in mice when administered immediately after ectopic U87MG xenograft implantation (54% decrease, P ≤ 0.05) or in mice with established xenografts (48% decrease, P ≤ 0.05) without any apparent toxicity. Importantly, AsA feeding (30 mg/kg/twice a day) also decreased the orthotopic U87MG xenografts growth in nude mice as measured by magnetic resonance imaging. Using LC/MS-MS methods, AsA was detected in mice plasma and brain tissue, confirming that AsA crosses blood-brain barrier. Mechanistic studies showed that AsA induces apoptotic death by modulating the protein expression of several apoptosis regulators (caspases, Bcl2 family members, and survivin) in GBM cells. Furthermore, AsA induced ER stress (increased GRP78 and Calpain, and decreased Calnexin and IRE1α expression), enhanced free intra-cellular calcium, and damaged cellular organization in GBM cells. These experimental results demonstrate that AsA is effective against GBM, and advocate further pre-clinical and clinical evaluations of AsA against GBM.

Topical nitrogen mustard exposure causes systemic toxic effects in mice.

Vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) are reported to be easily absorbed by skin upon exposure causing severe cutaneous injury and blistering. Our studies show that topical exposure of NM (3.2mg) onto SKH-1 hairless mouse skin, not only caused skin injury, but also led to significant body weight loss and 40-80% mortality (120 h post-exposure), suggesting its systemic effects. Accordingly, further studies herein show that NM exposure initiated an increase in circulating white blood cells by 24h (neutrophils, eosinophils and basophils) and thereafter a decrease (neutrophils, lymphocytes and monocytes). NM exposure also reduced both white and red pulp areas of the spleen. In the small intestine, NM exposure caused loss of membrane integrity of the surface epithelium, abnormal structure of glands and degeneration of villi. NM exposure also resulted in the dilation of glomerular capillaries of kidneys, and an increase in blood urea nitrogen/creatinine ratio. Our results here with NM are consistent with earlier reports that exposure to higher SM levels can cause damage to the hematopoietic system, and kidney, spleen and gastrointestinal tract toxicity. These outcomes will add to our understanding of the toxic effects of topical vesicant exposure, which might be helpful towards developing effective countermeasures against injuries from acute topical exposures.

Silibinin prevents prostate cancer cell-mediated differentiation of naïve fibroblasts into cancer-associated fibroblast phenotype by targeting TGF ß2.

Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) ß2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFß2-neutralizing antibody. The inhibitory effect of silibinin on TGFß2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM.

Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling.

Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 µM) treatment significantly decreased the fibronectin (5 µg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, ß1 and ß3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and ß-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells' interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression.