Pre-treatment of rapamycin transformed M2 microglia alleviates traumatic cervical spinal cord injury via AIM2 signaling pathway in vitro and in vivo.

BACKGROUND: Traumatic spinal cord injury (SCI) is still devastating. It was suggested that the inhibition of mTOR may alleviate neuronal inflammatory injury but its underlying mechanism remained to be elucidated. AIM2 (absent in melanoma 2) recruits ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses. We designed this study to elucidate whether pre-treatments of rapamycin could suppress SCI induced neuronal inflammatory injury via AIM2 signaling pathway in vitro and in vivo. METHODS: We performed oxygen and glucose deprivation / re-oxygenation (OGD) treatment and rats clipping model to mimic neuronal injury after SCI in vitro and in vivo. Morphologic changes of injured spinal cord were detected by hematoxylin and eosin staining. The expression of mTOR, p-mTOR, AIM2, ASC, Caspase-1 and et al were analyzed by fluorescent staining, western blotting or qPCR. The polarization phenotype of microglia was identified by flow cytometry or fluorescent staining. RESULTS: We found BV-2 microglia without any pre-treatment cannot alleviate primary cultured neuronal OGD injury. However, pre-treated rapamycin in BV-2 cells could transform microglia to M2 phenotype and protects against neuronal OGD injury via AIM2 signaling pathway. Similarly, pre-treatment of rapamycin could improve the outcome of cervical SCI rats through AIM2 signaling pathway. CONCLUSIONS: It was suggested that resting state microglia pre-treated by rapamycin could protect against neuronal injury via AIM2 signaling pathway in vitro and in vivo. Pre-inhibition of mTOR pathway may improve neuronal protection after SCI.

Blood Pressure Variability Associates with Six-Month Outcomes in Acute Cervical Spinal Cord Injury: An Analysis of 105 Patients.

OBJECTIVE: Blood pressure variability (BPV) has been shown to correlate with poor outcomes in patients with intracerebral hemorrhage (ICH) and traumatic brain injury. However, this association has not been elucidated in patients with traumatic cervical spinal cord injury (cSCI). We hypothesized that 24-hour BPV from time of admission is associated with worse outcomes in patients with cSCI. METHODS: We performed a retrospective chart review analysis of adult patients at Huashan Hospital Fudan University between January 2006 and September 2022. We included isolated patients with traumatic cSCI within 6 hours of injury. Outcomes of patients with cSCI were assessed using 6-month American Spinal Injury Association (ASIA) impairment scale grade, and were dichotomized into poor (ASIA grade A-C, or decreasing ASIA grade compared with baseline) and good (ASIA grade D and E, or increasing ASIA grade compared with baseline) outcome groups. Blood pressures (BPs) were recorded during the first 24 hours of hospital course. BP was analyzed in the hyperacute period, from 0 to 4-5 hours; and in the acute period, from 4-5 to 24-25 hours after admission. BPV was analyzed by standard deviation (SD), coefficient of variation (CV), and successive variation (SV) of systolic BP (SBP). RESULTS: We analyzed 105 patients' charts. The first BP assessment, on emergency department arrival, at median 267 minutes (interquartile range, 152-312 minutes) after onset of injury was mean 152.2 mm Hg (SD, 51.8 mm Hg). The second BP assessment, on neurosurgical intensive care unit arrival, was mean 148.1 mm Hg (53.2 mm Hg). Poor outcomes occurred in 63 patients (60%). In univariate analysis, univariate quintile analysis or multivariate analysis, SBPSD, SBPCV, and SBPSV were associated with poor outcomes in both the hyperacute and the acute period. CONCLUSIONS: BPV during the first 24 hours after injury in patients with traumatic cSCI was independently associated with poor functional outcome at 3 months. Stabilization of BPV during the hyperacute and acute period may be a therapeutic target to improve functional outcomes of these patients.

Realistic long-term dysphagia rates after anterior cervical discectomy with fusion: is there a correlation with postoperative sagittal alignment and lordosis at a minimum 2-year follow-up?

OBJECTIVE: Postoperative dysphagia after anterior cervical discectomy and fusion (ACDF) has many contributing factors, and long-term data are sparse. The authors evaluated dysphagia after ACDF based on levels fused and cervical sagittal parameters. METHODS: Patients who underwent ACDF between 2009 and 2018 at the University of California, San Francisco (UCSF), were retrospectively studied. Dysphagia was evaluated preoperatively, immediately postoperatively, and at last follow-up using the UCSF dysphagia score. Dysphagia was categorized as normal (level 7), mild (levels 5 and 6), moderate (levels 3 and 4), and severe (levels 1 and 2). The UCSF mild dysphagia score was further classified as "minimal dysphagia," while moderate and severe dysphagia were classified as "significant dysphagia." "Any dysphagia" included any dysphagia, regardless of grade. Cervical sagittal parameters were measured preoperatively, immediately postoperatively, and at last follow-up. RESULTS: A total of 131 patients met inclusion criteria. The mean follow-up was 43.89 (24-142) months. Seventy-eight patients (59.5%) reported dysphagia immediately postoperatively, and 44 patients (33.6%) reported some dysphagia at last follow-up (p < 0.001). The rates of moderate dysphagia were 13.0% immediately postoperatively and 1.5% at the last follow-up (p < 0.001). Twenty-two patients (16.8%) had significant dysphagia immediately postoperatively, and 2 patients (1.5%) had significant dysphagia at last follow-up (p < 0.001). Patients with immediate postoperative dysphagia had less C2-7 preoperative lordosis (-9.35°) compared with patients without (-14.15°, p = 0.029), but there was no association between C2-7 lordosis and dysphagia at last follow-up (p = 0.232). The prevalence rates of immediate postoperative dysphagia and long-term dysphagia were 87.5% and 58.3% in ≥ 3-level ACDF; 64.0% and 40.0% in 2-level ACDF; and 43.9% and 17.5% in 1-level ACDF, respectively (p < 0.001). CONCLUSIONS: The realistic incidence of any dysphagia after ACDF was 59.5% immediately postoperatively and 33.6% at the minimum 2-year follow-up, higher than previously published rates. However, most dysphagia was not severe. The number of fused levels was the most important risk factor for long-term dysphagia, but not for immediate postoperative dysphagia. Loss of preoperative C2-7 lordosis was associated with immediate postoperative dysphagia, but not long-term dysphagia. ACDF segmental lordosis and cervical sagittal vertical axis were not associated with long-term dysphagia in ACDF.

p53 Inhibition Protects against Neuronal Ischemia/Reperfusion Injury by the p53/PRAS40/mTOR Pathway.

The underlying mechanisms of cerebral ischemia/reperfusion (I/R) injury are unclear. Within this study, we aimed to explore whether p53 inhibition exerts protective effects via the p53/PRAS40/mTOR pathway after stroke and its potential mechanism. Both an in vitro oxygen-glucose deprivation (OGD) model with a primary neuronal culture and in vivo stroke models (dMCAO or MCAO) were used. We found that the infarction size, neuronal apoptosis, and autophagy were less severe in p53 KO mice and p53 KO neurons after cerebral I/R or OGD/R injury. By activating the mTOR pathway, p53 knockdown alleviated cerebral I/R injury both in vitro and in vivo. When PRAS40 was knocked out, the regulatory effects of p53 overexpression or knockdown against stroke disappeared. PRAS40 knockdown could inhibit the activities of the mTOR pathway; moreover, neuronal autophagy and apoptosis were exacerbated by PRAS40 knockdown. To sum up, in this study, we showed p53 inhibition protects against neuronal I/R injury after stroke via the p53/PRAS40/mTOR pathway, which is a novel and pivotal cerebral ischemic injury signaling pathway. The induction of neuronal autophagy and apoptosis by the p53/PRAS40/mTOR pathway may be the potential mechanism of this protective effect.

Does transforaminal lumbar interbody fusion induce lordosis or kyphosis? Radiographic evaluation with a minimum 2-year follow-up.

OBJECTIVE: Conflicting reports exist about whether transforaminal lumbar interbody fusion (TLIF) induces lordosis or kyphosis, ranging from decreasing lordosis by 3.71° to increasing it by 18.8°. In this study, the authors' aim was to identify factors that result in kyphosis or lordosis after TLIF. METHODS: A single-center, retrospective study of open TLIF without osteotomy for spondylolisthesis with a minimum 2-year follow-up was undertaken. Preoperative and postoperative clinical and radiographic parameters and cage specifics were collected. TLIFs were considered to be "lordosing" if postoperative induction of lordosis was > 0° and "kyphosing" if postoperative induction of lordosis was ≤ 0°. RESULTS: A total of 137 patients with an average follow-up of 52.5 months (range 24-130 months) were included. The overall postoperative disc angle (DA) and segmental lordosis (SL) increased by 1.96° and 1.88° (p = 0.003 and p = 0.038), respectively, whereas overall lumbar lordosis remained unchanged (p = 0.133). Seventy-nine patients had lordosing TLIFs with a mean SL increase of 5.72° ± 3.97°, and 58 patients had kyphosing TLIFs with a mean decrease of 3.02° ± 2.98°. Multivariate analysis showed that a lower preoperative DA, lower preoperative SL, and anterior cage placement were correlated with the greatest increase in postoperative SL (p = 0.040, p < 0.001, and p = 0.035, respectively). There was no difference in demographics, cage type or height, or spinopelvic parameters between the groups (p > 0.05). Linear regression showed that the preoperative DA and SL correlated with SL after TLIF (R2 = 0.198, p < 0.001; and R2 = 0.2931, p < 0.001, respectively). CONCLUSIONS: Whether a TLIF induces kyphosis or lordosis depends on the preoperative DA, preoperative SL, and cage position. Less-lordotic segments became more lordotic postoperatively, and highly lordotic segments may lose lordosis after TLIF. Cages placed more anteriorly were associated with more lordosis.

The Effect of Anterior Cervical Discectomy and Fusion on Cervical Sagittal Vertical Axis and Lordosis with Minimum 2-Year Follow-Up.

BACKGROUND: Anterior cervical discectomy and fusion (ACDF) can induce lordosis and improve cervical sagittal vertical axis (SVA), but multilevel ACDF may inadvertently increase cervical SVA because of insufficient lordosis induction. METHODS: Patients who underwent 1-, 2-, or ≥3-level ACDF in the subaxial spine with minimum 2-year follow up were retrospectively studied. C2-C7 Cobb angle (lordosis), cervical SVA, and T1 slope were measured preoperatively, immediately postoperatively, and at last follow-up. RESULTS: Inclusion criteria were met by 127 patients. There were no differences in baseline demographics among 1-, 2-, and ≥3-level ACDF groups. Mean follow-up was 43.7 months (range, 24-142 months). Increase of cervical SVA immediately postoperatively was 1.94 mm, -1.44 mm, and 7.25 mm for 1-, 2-, and ≥3-level ACDF (P = 0.041) and at last follow-up was 2.97 mm, 0.70 mm, and 9.32 mm for 1-, 2-, and ≥3-level ACDF (P = 0.026). At last follow-up, 2-level ACDF patients had the greatest decrease in T1 slope (-0.43°) compared with increase of 2.71° for 1-level and 2.84° for ≥3-level patients (P = 0.028). In all 3 groups, segmental (ACDF levels) lordosis, cervical SVA, and T1 slope did not decrease from immediate postoperative to last follow-up. Only 2-level ACDF maintained C2-7 lordosis (2.16°) compared with loss of lordosis in 1-level (-0.84°) and ≥3-level (-2.00°) ACDF (P = 0.008) at last follow-up. Linear regression analysis showed that T1 slope had no relationship with correction of cervical SVA (P = 0.5310) but had a significant correlation with Cobb angle loss of C2-C7 lordosis (P = 0.0016). CONCLUSIONS: Compared with 1- and 2-level ACDF, ≥3-level ACDF resulted in significant increase of cervical SVA and loss of overall lordosis. Compared with 1- and ≥3-level ACDF, 2-level ACDF had the greatest ability to maintain lordosis. T1 slope had a significant correlation with loss of C2-C7 lordosis after ACDF.

Expression of TNF­α and IL­ß can be suppressed via the PPAR­Î³/mTOR signaling pathway in BV­2 microglia: A potential anti­inflammation mechanism.

Currently, microglia are considered as crucial factors in suppressing inflammatory reactions, but the specific molecular mechanism remains unknown. To elucidate whether peroxisome proliferator­activated receptor­Î³ (PPAR­Î³) can inhibit neuroinflammatory cytokine expression via the mTOR signal pathway, the BV­2 cell line was incubated with lipopolysaccharide (10 mM/ml) to induce an inflammatory injury. PPAR­Î³ was activated by rosiglitazone, and was inhibited by GW9662. The mTOR signal pathway was activated by phosphatidic acid (P.A.), while it was inhibited by rapamycin. Western blotting and reverse transcription­quantitative PCR were used to evaluate the expression levels of PPAR­Î³/mTOR signal pathway related proteins and neuroinflammatory cytokines, including NF­κB, tumor necrosis factor (TNF)­α and interleukin (IL)­1ß. When treated with P.A., the expression levels of phosphorylated (p)mTOR and p­ribosomal protein S6 kinase (pS6K) were significantly increased and the expression levels of TNF­α and IL­1ß were significantly lower. However, the expression of PPAR­Î³ was similar in P.A. treated cells and cells treated with rapamycin. When PPAR­Î³ was activated, pmTOR and pS6K protein expression levels were significantly decreased, and the mRNA expression levels of TNF­α and IL­1ß were significantly reduced, but this inhibition could be alleviated by administrating GW9662. Collectively, it was indicated that the mTOR signal pathway may be located downstream of PPAR­Î³. Furthermore, neuroinflammatory reactions could be inhibited via the activation of PPAR­Î³ by suppressing the mTOR signal pathway in microglia.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin.

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

Involvement of ROS-p38-H2AX axis in novel curcumin analogues-induced apoptosis in breast cancer cells.

Curcumin-based structural modification for developing more effective curcumin analogues has been drawning increasing attention. As alternative approach, using LC/MS guided purification, we previously obtained a series of novel natural terpene-conjugated curcuminoids from turmeric, and some of them exhibited even more potent anti-cancer activity against multiple types of cancer cells than curcumin. The purpose of this follow-up study was designed to decipher the mechanisms involved in anti-cancer activity of these novel curcumin analogues. Apoptosis was evaluated using sub-G1 analysis by flow cytometry and Cell Death ELISA Kit. Changes of protein expression were analyzed by western blotting. RNA interference was employed to inhibit expression of specific protein. We found that bisabolocurcumin ether (T1) and demethoxybisabolocurcumin ether (T2) were able to trigger much stronger apoptosis induction in multiple types of cancer cells than curcumin, which was attributed to persistent and stronger ROS generation. ROS induction by T1 resulted in activation of p38/H2AX axis and p53. Inhibition of p38/H2AX led to a significant reduction of apoptosis, whereas inactivation of p53 caused a dramatically enhanced H2AX phosphorylation and apoptosis induction, suggesting activation of p38/H2AX contributed to apoptosis induction by T1, whereas p53 activation protected novel curcumins-induced apoptosis via suppression of H2AX activation. Our findings provide mechanistic support for the potential use of terpene-conjugated curcuminoids as a novel class of cancer chemopreventive agents. © 2015 Wiley Periodicals, Inc.

Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-ß-D-glucose-induced senescence-like growth arrest in human cancer cells.

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-ß-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.

Density functional theory calculations in stereochemical determination of terpecurcumins J-W, cytotoxic terpene-conjugated curcuminoids from Curcuma longa L.

Fourteen novel terpene-conjugated curcuminoids, terpecurcumins J-W (1-14), have been isolated from the rhizomes of Curcuma longa L. Among them, terpecurcumins J-Q and V represent four unprecedented skeletons featuring an unusual core of hydrobenzannulated[6,6]-spiroketal (1 and 2), bicyclo[2.2.2]octene (3-7), bicyclo[3.1.3]octene (8), and spiroepoxide (13), respectively. The structures of compounds 1-14 were elucidated by extensive spectroscopic analysis, and their absolute configurations were established by electronic circular dichroism, vibrational circular dichroism, and (13)C NMR spectroscopic data analysis, together with density functional theory calculations. The structure and configuration of 1 was further confirmed by single-crystal X-ray diffraction (Cu Kα). The biogenetic pathways of 1-14 were proposed, involving Michael addition, condensation, Diels-Alder cycloaddition, and electrophilic substitution reactions. Terpecurcumins showed more potent cytotoxic activities than curcumin and ar-/ß-turmerone. Among them, terpecurcumin Q (8) exhibited IC50 of 3.9 µM against MCF-7 human breast cancer cells, and mitochondria-mediated apoptosis played an important role in the overall growth inhibition. Finally, LC/MS/MS quantitative analysis of five representative terpecurcumins indicated these novel compounds were present in C. longa at parts per million level.

Involvement of unfolded protein response, p53 and Akt in modulation of porcine reproductive and respiratory syndrome virus-mediated JNK activation.

Our previous study has shown that activation of JNK plays a critical role in Porcine reproductive and respiratory syndrome virus (PRRSV)-mediated apoptosis. In this follow-up study, we further investigated the mechanisms involved in modulation of PRRSV-mediated JNK activation and apoptosis. We found that unfolded protein response (UPR) was induced in response to PRRSV infection which in turn triggered JNK activation and apoptosis. We also found that p53 and Akt were activated at the early stage of infection and functioned as negative regulator of JNK activation to counteract the PRRSV-mediated apoptosis. Furthermore, induction of UPR, p53 and Akt was not only involved in modulation of PRRSV-mediated apoptosis, but also contributed to the virus replication. Our findings indicated that multiple signaling pathways were involved in modulation of PRRSV-mediated apoptosis of the host cells via regulating JNK signaling pathway and provided novel insights into understanding the mechanisms of pathogenesis of PRRSV infection.

Pseudolaric acid B induces caspase-dependent apoptosis and autophagic cell death in prostate cancer cells.

Previous studies have shown that Pseudolaric acid B (PAB), a medicinal plant-derived terpenoid, is a promising chemopreventive or therapeutic agent against various types of cancer. However, less is known regarding its activity against prostate cancer. The objective of the current study is designed to determine the anti-prostate cancer effects of PAB. We demonstrated here that PAB was highly effective against both androgen-dependent (LNCaP) and androgen-independent prostate cancer cells (PC-3 and DU145) through the mechanisms involved in induction of G2/M cell cycle arrest, caspase-dependent apoptosis and autophagic cell death. Furthermore, PAB can greatly sensitize these two aggressive androgen-independent prostate cancer cells to ABT-737-induced cytotoxicity. Our findings suggest that PAB holds excellent potential as either novel chemopreventive agent or enhancer of therapeutic drug against prostate cancer.

Enhanced apoptotic effects by the combination of curcumin and methylseleninic acid: potential role of Mcl-1 and FAK.

Curcumin and methylseleninic acid (MSeA) are well-documented dietary chemopreventive agents. Apoptosis appears to be a major mechanism for both agents to exert anti-cancer activity. The purpose of the present study was designed to determine whether the apoptotic effect on human cancer cells can be enhanced by combining curcumin with MSeA. Apoptosis was evaluated by Annexin V staining of externalized phosphatidylserine by flow cytometry. Expression of protein was analyzed by Western blotting. Localization of apoptosis-inducing factor (AIF) was detected by immunocytochemistry. RNA interference was employed to inhibit expression of specific protein. We found here that combining curcumin with MSeA led to a significantly enhanced apoptosis in both MDA-MB-231 breast cancer cells and DU145 prostate cancer cells. Further mechanistic investigations revealed that curcumin treatment alone caused a concentration dependent upregulation of Mcl-1, which can be overcome by combining it with MSeA. In line with the Mcl-1 reduction, an enhanced mitochondrial permeability transition and AIF nuclear translocation by the combination were achieved. In addition, an increased suppression of focal adhesion kinase activity was observed in the combination-treated cells which were associated with cell detachment-induced apoptosis by the combination. Our findings suggest that curcumin/MSeA combination holds excellent potential for improving their efficacy against human breast and prostate cancer through enhanced apoptosis induction.

Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells.

Ursolic acid (UA) is a pentacyclic triterpenoid with promising cancer chemopreventive properties. A better understanding of the mechanisms underlying anticancer activity of UA is needed for further development as a clinically useful chemopreventive agent. Here, we found that both endoplasmic reticulum (ER) stress and autophagy were induced by UA in MCF-7 human breast cancer cells. Surprisingly, ER stress was identified as an effect rather than a cause of UA-induced autophagy. Autophagy-dependent ER stress protected the cells from UA-induced apoptosis through EIF2AK3-mediated upregulation of MCL1. Activation of MAPK1/3 but not inhibition of MTOR pathway contributed to UA-induced cytoprotective autophagy in MCF-7 cells. Our findings uncovered a novel cellular mechanism involved in the anticancer activity of UA, and also provided a useful model to study biological significance and mechanisms of autophagy-mediated ER stress.

Terpecurcumins A-I from the rhizomes of Curcuma longa: absolute configuration and cytotoxic activity.

Terpecurcumins A-I (1-9), together with three known analogues (10-12), were isolated from the rhizomes of Curcuma longa (turmeric). They were derived from the hybridization of curcuminoids and bisabolanes. The structures and absolute configurations of 1-9 were elucidated on the basis of extensive spectroscopic data analysis, including NMR and electronic circular dichroism spectra. The configuration of 10 was further confirmed by X-ray crystallography. A plausible biogenetic relationship for 1-12 is proposed. Compounds 4, 6, 7, 10, and 11 showed higher cytotoxic activities (IC(50), 10.3-19.4 µM) than curcumin (IC(50), 31.3-49.2 µM) against human cancer cell lines (A549, HepG2, and MDA-MB-231).

Activation of c-Jun NH(2)-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication.

Apoptosis of host cells plays a critical role in pathogenesis of virus infection. MAPK kinases especially stress-activated protein kinases c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 are often involved in virus-mediated apoptosis. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) infection resulted in apoptosis of the host cells both in vitro and in vivo. The current investigation was initiated to determine whether stress-activated protein kinases JNK and p38 play a role in apoptosis induction by PRRSV infection. We examined phosphorylation of JNK and p38, and found that JNK but not p38 was activated in response to PRRSV infection. We then examined effects of this kinase on apoptosis induction and virus replication by using specific inhibitor. We found that JNK inhibition by its inhibitor SP600125 led to the abolishment of PRRSV-mediated apoptosis, but did not suppress virus replication. Further studies demonstrated that ROS generation was involved in JNK activation, and Bcl-2 family anti-apoptotic proteins Mcl-1 and Bcl-xl were downstream targets of JNK to mediate apoptosis. We conclude that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not for virus replication.

Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad.

ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can overcome such resistance to restore the sensitivity. In the present study, a second-generation selenium compound methylseleninic acid (MSeA) synergistically sensitized MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells and DU145 human prostate cancer cells to apoptosis induction by ABT-737, as evidenced by greater than additive enhancement of Annexin V/FITC positive (apoptotic) cells and activation of multiple caspases and PARP cleavage. Mechanistic investigation demonstrated that MSeA significantly decreased basal Mcl-1 expression and ABT-737-induced Mcl-1 expression. Knocking down of Mcl-1 with RNAi approach supported the functional significance of this molecular target. More importantly, we identified inactivation of Bad by phosphorylation on ser-136 and ser-112 as a novel mechanism involved in ABT-737 resistance, which can be overcome by combining with MSeA. In addition, we found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737.

p53 activation inhibits ochratoxin A-induced apoptosis in monkey and human kidney epithelial cells via suppression of JNK activation.

Ochratoxin A (OTA), one of the major food-borne mycotoxins, induces apoptosis in various types of cells. Induction of apoptosis is suggested to be one of the major cellular mechanisms behind OTA-induced diverse toxic effects. However, the molecular mechanisms involved, especially the role of p53 in OTA-induced apoptosis have not been clearly elucidated. In the present study, we find that p53 activation exerts pro-survival function to inhibit apoptosis induction in MARC-145, Vero monkey kidney cells and HEK293 human kidney cells in response to ochratoxin A treatment. We further demonstrate that the pro-survival activity of p53 is attributed to its ability to suppress JNK activation that mediates apoptotic signaling through down-regulation of Bcl-xL. To our knowledge, this is first report of pro-survival role of p53 in OTA-induced apoptosis in kidney epithelial cells. Our findings provide a novel insight into the mechanisms of OTA-induced apoptosis in kidney epithelial cells.

Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata.

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-ß-D-glucoside (1c), 1ß-hydroxybufotalin (1d), and 5ß-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ¹-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.