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Supplementary Figure from A Randomized, Open-Label, Multicenter, Phase 3 Study of High-Dose Vitamin C Plus FOLFOX ± Bevacizumab versus FOLFOX ± Bevacizumab in Unresectable Untreated Metastatic Colorectal Cancer (VITALITY Study)

Supplementary Data from A Randomized, Open-Label, Multicenter, Phase 3 Study of High-Dose Vitamin C Plus FOLFOX ± Bevacizumab versus FOLFOX ± Bevacizumab in Unresectable Untreated Metastatic Colorectal Cancer (VITALITY Study)

ARX788 is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugate with AS269 as cytotoxic payload. In this phase 1 multicenter dose-expansion clinical trial, patients with HER2-positive advanced gastric/gastroesophageal junction adenocarcinoma failing to respond to prior trastuzumab-based standard treatment were enrolled. Between July 15

Purpose: To compare the efficacy and safety of high-dose vitamin C plus FOLFOX ± bevacizumab versus FOLFOX ± bevacizumab as first-line treatment in patients with metastatic colorectal cancer (mCRC). Patients and Methods: Between 2017 and 2019, histologically confirmed patients with mCRC (n = 442) with normal glucose-6-phosphate dehydrogenase status and no prior treatment for metastatic disease were randomized (1:1) into a control (FOLFOX ± bevacizumab) and an experimental [high-dose vitamin C (1.5 g/kg/d, intravenously for 3 hours from D1 to D3) plus FOLFOX ± bevacizumab] group. Randomization was based on the primary tumor location and bevacizumab prescription. Results: The progression-free survival (PFS) of the experimental group was not superior to the control group [median PFS, 8.6 vs. 8.3 months; HR, 0.86; 95% confidence interval (CI), 0.70–1.05; P = 0.1]. The objective response rate (ORR) and overall survival (OS) of the experimental and control groups were similar (ORR, 44.3% vs. 42.1%; P = 0.9; median OS, 20.7 vs. 19.7 months; P = 0.7). Grade 3 or higher treatment-related adverse events occurred in 33.5% and 30.3% of patients in the experimental and control groups, respectively. In prespecified subgroup analyses, patients with RAS mutation had significantly longer PFS (median PFS, 9.2 vs. 7.8 months; HR, 0.67; 95% CI, 0.50–0.91; P = 0.01) with vitamin C added to chemotherapy than with chemotherapy only. Conclusions: High-dose vitamin C plus chemotherapy failed to show superior PFS compared with chemotherapy in patients with mCRC as first-line treatment but may be beneficial in patients with mCRC harboring RAS mutation.

Esophageal cancer is one of the most common malignant digestive diseases worldwide. Although many approaches have been established for the treatment of esophageal cancer, the survival outcome has not improved. Pristimerin is a quinone methide triterpenoid with anticancer, antiangiogenic, anti-inflammatory, and antiprotozoal activities. However, the role of pristimerin in cancers such as esophageal cancer is unclear. In this study, we investigated the role and mechanisms of action of pristimerin in esophageal cancer. First, we found that pristimerin can induce apoptosis in esophageal cancer in vivo and in vitro. CCK-8 and clonogenic assays showed that pristimerin decreased the growth of Eca109 cells. In addition, we found that pristimerin decreased the protein expression of CDK2, CDK4, cyclin E, and BCL-2 and increased the expression of CDKN1B. Meanwhile, pristimerin elevated the ratio of LC3-II/LC3-I. Otherwise, downregulation of CDKN1B can reduce the esophageal cancer tumor growth induced by pristimerin. In conclusion, our findings revealed an important role of pristimerin in esophageal cancer and suggest that pristimerin might be a potential therapeutic agent for this cancer.

e15061 Background: There are limited therapeutic options for the treatment of advanced colorectal cancer which fail first-line chemotherapy. Phase I studies have shown that the combined application of the irinotecan (CPT-11) and raltitrexed has significant synergistic effect and acceptable toxicity. The aim of this multicenter study was to assess the efficacy and toxicity of second-line raltitrexed plus irinotecan in Chinese patients with advanced colorectal cancer. Methods: This is an open-label,single-arm, multicenter, phase II trial (Registered in clinicaltrials.gov with NCT03053167).Brief inclusion criteria: patients were aged 18 to 75 years with locally advanced or metastatic colorectal cancer after failure of oxaliplatin and fluorouracil therapy. Enrolled patients received CPT-11 (180 mg /m2 , d1) and raltitrexed (3 mg/m2, d1) each 21-day cycle until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were disease control rate (DCR), objective response rate (ORR), overall survival (OS), quality of life (QOL) and safety. In all, 100 patients were required for primary point testing. Results: Between November 2016 and December 2018, a total of 70 patients were screened for enrollment and 53 patients reached the primary endpoint. Nine patients achieved a partial response and twenty-seven stable disease. The overall response rate was 17% (9/53) and the disease control rate was 67.9% (36/53). Median progression-free survival (mPFS) was 4.3 months and median overall survival was not observed. The most common adverse events were elevated transaminases (21/53), fatigue (14/53), diarrhea (12/53), neutrocytopenia (10/53), erythrocytopenia (9/53), hypohemoglobin (8/53) and leukocytopenia (6/53). The total incidence of grade 3/4 toxicity was 17% (9/53) , mainly diarrhea (2/53), neutrocytopenia (2/53) and elevated transaminases (2/53). There were no treatment-related deaths. Conclusions: We have demonstrated that CPT-11 plus raltitrexed is active and feasible in patients with second-line treatment in advanced colorectal cancer. This trial will progress as planned. Clinical trial information: NCT03053167.

The incidence of Parkinson's disease (PD) is increasing as the global population ages. 6-hydroxydopamine (6-OHDA) can induce PD-like neuropathology and biochemical changes in both in vitro and in vivo models. Therefore, clarification of the molecular mechanism of 6-OHDA-induced cell death might contribute to the understanding of the pathogenesis of PD.With this goal in mind, we investigated the role of protein kinase C delta (PKC delta) in 6-OHDA-dependent death using the pheochromocytoma cell line, PC12. Cells were treated with 6-OHDA to induce toxicity with or without pretreatment using rottlerin (a PKC delta inhibitor), bisindolylmaleimide I (a general PKC inhibitor), Gö6976 (a PKC inhibitor selective for calcium-dependent PKC isoforms), or phorbol-12-myristate-13-acetate (PMA, a PKC activator).Phorbol-12-myristate-13-acetate decreased cell survival and increased the rate of apoptosis while rottlerin increased cell survival and decreased the rate of apoptosis. In contrast, neither bisindolylmaleimide I nor Gö6976 affected 6-OHDA-induced cell death. Western analysis demonstrated that phosphorylation of PKC delta on Thr 505 as well as extracellular signal-regulated kinase (ERK) phosphorylation increased after exposure to 6-OHDA. This increase in PKC delta phosphorylation was potentiated by PMA. However, rottlerin attenuated the 6-OHDA-stimulated increase in PKC delta and ERK phosphorylation.These data suggest that PKC delta, rather than classic-type PKC (alpha, beta1, beta2, gamma), participates in 6-OHDA-induced neurotoxicity in PC12 cells, and PKC delta activity is required for subsequent ERK activation during cell death.

The clinical side-effects of increased cyclooxygenase (COX) activity induced by pathologic conditions have raised concerns recently. However, a better understanding of the mechanisms underlying the subsequent neurotoxicity requires knowledge of pathways downstream of COX, especially prostaglandin E2 (PGE2) and its receptors. Therefore, this study was performed to investigate the effects of PGE2 receptor 1 (EP1) activity on neuronal cell death resulting from hypoxia/reoxygenation (Hyp). As cyclinD1 activity has been shown to regulate neuronal apoptosis as well, the role of cyclinD1 was investigated, as well. Cortical neural cells isolated from fetal Wistar rats were cultured for 12 d and exposed to Hyp conditions to establish an in vitro Hyp model. To determine the effects of EP1 activity on Hyp-induced neurotoxicity, cells were treated with 17-phenyl trinor-PGE2 (17-pt), a synthetic EP1 agonist, or sc-51089, an EP1 antagonist, then exposed to hypoxic conditions for 3h and reoxygenated for 21 h. Following Hyp, cell viability was quantified by MTT assays, and apoptosis was assessed by flow cytometry. Protein expression levels of caspase-3 and cyclinD1 were examined by Western blot analysis. Treatment of cultured cortical neurons with 17-pt significantly decreased the survival rate of Hyp-treated neurons (p0.05), while treatment with sc-51089 increased the survival rate. Treatment with 17-pt also led to increased expression levels of caspase-3, further supporting a role for EP1 in the observed neurotoxicity. However, cyclinD1 expression levels were unchanged following treatment with either 17-pt or sc-51089. Therefore, EP1 may play an important role in Hyp-induced neuronal apoptosis, but this neurotoxic activity is unlikely to involve cyclinD1.

To explore the clinical value and safety of TRUS-guided transperineal biopsy with the 9 + X method in the diagnosis of prostate carcinoma.A total of 420 men underwent TRUS-guided transperineal biopsy with the 9 + X method for suspected prostate carcinoma. Their clinical data were retrospectively analyzed.Prostate carcinoma was detected in 160 (38.1%) of the 420 cases, accounting for 7.4%, 17.8% and 65.4% in those with PSA4.0 microg/L, 4 -10 microg/L and10 microg/L respectively, 25.0% in those with abnormal findings on digital rectal examination (DRE), and 22.2% in those with abnormal echoes on TRUS or abdominal ultrasound examination. Complications after prostatic biopsy included gross hematuria in 79 cases (18.8%), acute urinary retention in 13 (3.1%) and fever in 9 (2.1%), but no other serious complications were observed.TRUS-guided transperineal biopsy with the 9 + X method, with high accuracy and fewer complications, is an ideal approach to the diagnosis of prostate carcinoma.

To explore the diagnosis and treatment of primary epididymal tumor.We retrospectively analyzed the clinical data of 35 cases of pathologically confirmed primary epididymal tumor. Of the total number of patients, 10 underwent tumor excision, 23 received epididymectomy, 1 was treated by simple orchidoepididymectomy, and by radical orchidoepididymectomy with second-stage retroperitoneal lymph node dissection.Postoperative pathology confirmed 33 cases of benign tumor (including 21 adenomatoid tumor, 7 leiomyoma, 4 fibroma, and 1 papillary cystadenoma), and 2 cases of malignancy (1 malignant fibrous histiocytoma and 1 adenocarcinoma). The follow-up lasted 10 months to 6 years, which revealed no recurrence, metastasis and death.Primary epididymal tumor is difficult to be definitely diagnosed preoperatively. Surgical exploration is the first choice for those highly suspected of the disease. Tumor excision or epididymectomy can be considered for benign cases, while radical orchidoepididymectomy with retroperitoneal lymph node dissection is recommended in case of malignancy.

We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.

To investigate the role of phosphorylation of protein kinase C (PKC) delta in the toxicity of 6-hydroxydopamine (6-OHDA) to the death of dopaminergic neurons.Human neuroblastoma cells of the line SH-SY5Y were cultured 6-OHDA of the concentrations of 0, 50, 100, 200, and 400 micromol/L was added to observe its toxicity. Rottlerin (PKCdelta inhibitor, 2 micromol/L), bisindolylmaleimide (Bis, general PKC inhibitor, 10 nmol/L), Gö6976 (calcium-dependent PKC inhibitor, 5 nmol/L), and phobol-12-myristate-13-acetate (PMA, PKC activator, 100 nmol/L) were added into the culture fluid of another SH-SY5Y cells respectively, and then (1) culture fluid of equal volume was added for 18 h so as to observe there effects on the survival of the SH-SY5Y cells, or (2) 100 micromol/L 6-OHDA was added to observe the effects of intervention on PKC on the survival of the SH-SY5Y cells by using MTT assay. Cell lysis solution with phosphatase inhibitor was used to lyse the culture cells to extract plasma protein. Western blotting was used to detect the expression of phosphorylated PKCdelta.MTT assay showed that all different concentrations (50 - 400 micromol/L) of 6-OHDA significantly and dose-dependently caused cell death with an EC50 of 92 micromol/L. Pretreatment with rottlerin and Bis alone did not influence the survival of the cells significantly,. However, the survival rate of the cells pretreated by Gö6976 alone was 92.3% +/- 3.2% that of the control group (P0.01), and the survival rate of the cells pretreated by PMA was 49.5% +/- 1.0% that of the control group (P0.01) Pretreatment of rottlerin decreased the death rate of the cells treated with 6-OHDA to 30.4% +/- 1.6% and conferred significant protection against 6-OHDA neurotoxicity by 57% +/- 6% compared to that of the cells treated by 6-OHDA alone (P0.01). However, Bis and Gö6976 did not affect the 6-OHDA-induced cell damage. Pretreatment of PMA increased the death rate of the cells treated with 6-OHDA to 67.1% +/- 2.2% and significantly aggravated 6-OHDA-induced cell toxicity by 66% +/- 9% (P0.01). Western blotting showed that 6-OHDA administration increased the expression of phosphorylated PKCdelta, pretreatment with Rottlerin inhibited such increase, PMA promoted such increase, and Bis and Gö6976 did not influence such increase.Inhibition of PKCdelta phosphorylation with rottlerin ameliorates the neurotoxicity evoked by 6-OHDA, and activation of PKCdelta phosphorylation by PMA aggravates neurotoxicity, which implicating that this kinase participates in the 6-OHDA-induced neurotoxicity and Parkinsonian neurodegeneration.

Polyamines (putrescine, spermidine, and spermine) are present in all higher eukaryotic cells and are essential for cell growth, differentiation and apoptosis. Sharing common precursor with polyamines, nitric oxide (NO) is associated with myocardial ischemia/reperfusion injury by the generation of peroxynitrite. Although polyamines have been implicated in tissue ischemia injury, their metabolism and interactions with NO in myocardial ischemia/reperfusion injury have not been fully understood. In our experiment, when Langendorff perfused rat hearts were subjected to 40 min ischemia without reperfusion, both ornithine decarboxylase (ODC) and Spermidine/spermine N(1)-acetyltransferase (SSAT) activities were up-regulated and putrescine accumulated. While after reperfusion, ODC activity decreased and SSAT activity increased, resulting in putrescine accumulation and decreased spermidine and spermine. Meanwhile NO content was increased. In addition, sodium nitroprusside (SNP, a NO donor) decreased ODC activity in cardiac tissue homogenate but increased SSAT activity in a dose-dependent manner. Pre-treatment of isolated heart with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NO synthase) increased ODC activity. Exogenous spermine (1 mM) administration prior to ischemia prevented spermine decrease, reduced cardiac myocyte necrosis and apoptosis, and promoted the recovery of cardiac function after ischemia/reperfusion. These results suggest that acute heart ischemia activates myocardial polyamine stress response characterized by increased ODC and SSAT activities and accumulation of putrescine. Ischemia/reperfusion disturbs polyamine metabolism, and the loss of spermine might be associated with NO increase and thereby influences myocardial cell viability. Exogenous spermine may protect the hearts from myocardial ischemia/reperfusion injury.

To investigate the relationship between calcium-sensing receptor protein (CaSR) expression and rat cardiomyocyte apoptosis and related signal transduction pathways.The CaSR, BCl2, Caspase3 protein and ERK1/2 phosphorylation or non-phosphorylation were detected by Western blot. Cardiomyocyte apoptosis was detected by flow cytometry and immunofluorescence.CaSR protein was detected in rat cardiac tissue and CaSR activator gadolinium (GdCl3) induced cardiomyocyte apoptosis and increased ERK1/2 phosphorylation and expression of BCl2 and activated Caspase3. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished gadolinium -induced ERK1/2 activation and BCl2 expression, further increased the activation of Caspase3 and cardiomyocyte apoptosis.Our results demonstrate the CaSR existence in cardiomyocytes and CaSR activation by gadolinium can induce myocyte apoptosis by activating Caspase3 and tyrosine protein kinase pathway.

To investigate the relaxative characteristics of resveratrol on thoracic aortic artery in the rat and its mechanism.We perfused the isolated rings and observed the response of NA-induced artery contraction to resveratrol under the Ca2+-contained and Ca2+-free bath solutions. In the same way were the effect of reveratrol on the vascular smooth muscle observed by adding two different concentration of KCl (30 and 80 mmol x L(-1)), and the effect on the contraction of the vascular smooth muscle depending on the intracellular calcium and extracellular calcium were also observed by adding NA. We also observed the effect of resveratrol on the contraction of rings induced by NA in the presence of L-NNA and Glibenclamide.Resveratrol relaxed rat aorta rings precontracted by NA in a dose-dependent manner. The relaxant effect of resveratrol on the rat rings of endothelium-denuded group was reduced compared with that of endothelium-intact group; the relaxant effect of resveratrol on rat rings was higher under the condition of Ca2+-free bath solution than that under the condition of Ca2+-contained bath solution. Resveratrol had a repressive effect on the aorta's contraction induced by intracellular calcium, but had no effect induced by extracellular calcium. Resveratrol relaxed the contractions induced by KCl 30 mmol x L(-1) as well as KCl 80 mmol x L(-1), but the contraction curve of KCl 80 mmol x L(-1) was shifted upward significantly. In the L-NNA group, the relaxant effect was attenuated by (26.0 +/- 4.6) %; but there was no change in the group of Glibenclamide ( P0.05).The results indicate that resveratrol relaxes vascular smooth muscle in an endothelium dependent manner. The mechanisms for this phenomenon seem to be related with promoting synthesis and release of NO, opening Ca2+ activated K+ channel (KCa channel) as well as the inhibition of Ca2+ influx and release of Ca2+ from intracellular stores.

To observe the effects of peroxisome proliferation activated receptor (PPAR)-gamma on neuronal cell death induced by hypoxia/reoxygenation and ischemia/reperfusion.Cortical neural cells of fetal SD rats were cultured for 12 days and exposed to hypoxia/reoxygenation so as to establish a hypoxia/reoxygenation model. Another primary fetal rat cortical neuronal cells were pre-treated with different concentrations of GW9662, antagonist of PPAR-gamma, then underwent hypoxia for 3 hours, re-oxygenated for 21 hours. MTT was added one hour after to measure the cell viability. Eleven male SD rats underwent right middle cerebral artery occlusion (MCAO) using suture and reperfusion. Eleven rats underwent sham operation. after the rats were killed and their brains were taken out. Nucleoprotein was extracted from the cultured primary cortical cells and the cerebral cortexes of the rats and co-cultured with [gamma-(32)P]-labeled PPAR-gamma probe, EMSA to detect the PPAR-gamma binding activity.The PPAR-gamma activity of the cultured fetal rat cortical neurons that underwent hypoxia/reoxygenation significantly increased: It began to increase 1 hour after hypoxia and peaked in the 3rd hour of hypoxia, when the neurons underwent hypoxia for 3 hours and were reoxygenated for 2 hours, the binding activity still remained at a high level, and basically returned to the level of the untreated group 8 hours after reoxygenation. Data were quantified with control group as 100, 3 h of hypoxia was 160.3, and 2, 4, 8 h after reoxygenation were 157.5, 136.6, 103.3 separately. One hour after reperfusion the PPAR-gamma binding activity of the cortical cells at the ischemic side of the rats began to increase and peaked at the 4th hour, significantly higher than those of the cortical cells at the opposite side and of the sham operation group (both P0.01) then remained at a high level for the following 24 hours. The survival rate of the cultured neurons that underwent hypoxia for 3 hours and reoxygenation for 21 hours was significantly lower than that of the untreated neurons. Data were quantified with sham surgery group as 100, the side of MCAO and the contra side in surgery group were 144.8 and 102.6 separately. The survival rate of the neurons that were pretreated with GW9662 and then underwent hypoxia/reoxygenation was significantly higher than that of those without pretreatment (P0.01) with the peak protection effect of GW9662 at the concentration of 2.5 - 10 micromol/L. Data were quantified with control group as 100, hypoxia/reoxygenation group was 184, GW9662 group was 105. The PPAR-gamma binding activity of the primary cortical neurons pretreated with 5 micromol/L GW9662 for 30 minutes and than exposed to hypoxia for 3 hours and reoxygenation for 2 hours was significantly lower than that of the only hypoxia/reoxygenation group (P0.01).PPAR-gamma is involved in the pathogenesis of neuron death induced by hypoxia/ischemia and may become a new target of treatment of ischemic stroke.

The asymmetric unit of the title compound, C12H12N4O2, contains two independent mol­ecules. The dihedral angles between the planar rings of the two mol­ecules are 7.72 (3) and 40.24 (2)°. In the crystal structure, intra­molecular O—H⋯O and inter­molecular O—H⋯O, O—H⋯N and N—H⋯N hydrogen bonds form a network structure.