Exposure to zinc induces lysosomal-mitochondrial axis-mediated apoptosis in PK-15 cells.

Zinc (Zn), a kind of metallic element, can cause poisonous effects on host physiology when its excess exposure. Lysosomes and mitochondria are the toxic targets of heavy metals, and the lysosomal-mitochondrial axis is also verified to take part in apoptosis, but the related underlying mechanisms in Zn-induced cytotoxicity remain undefined. Here, we identified that excess Zn could cause cell damage in PK-15 cells accompanied by the lysosomal and mitochondrial dysfunction, with the evidence by the elevated levels of cathepsin B/D (CTSB/CTSD) in cytoplasm and decrease of Lyso-Tracker Red signal, red fluorescence intensity of AO staining, mitochondrial complex enzyme activities and ATP production. Additionally, the number of Annexin V+/PI--stained cells, apoptosis-related genes (Bax, Bid, Bak1, Caspase-9, and Caspase-3) and proteins levels of Bax, Bak1, Caspase-9, cleaved Caspase-3 and cytoplasmic Cyt C were signally elevated under Zn exposure, while the protein levels of Bcl2 and mitochondrial Cyt C were observably decreased. Importantly, Pepstatin A (the activity inhibitor of CTSD) and RNA interference of CTSD (si-CTSD) was used to reduce the release of lysosomal CTSD to the cytoplasm, which could signally alleviated Zn-induced mitochondrial damage and apoptosis. In summary, these results suggested that Zn could induced lysosomal and mitochondrial dysfunction in PK-15 cells, and the CTSD played an important role in Zn-induced lysosomal-mitochondrial axis-mediated apoptosis. Our results provided a new insight in Zn-induced toxicology, which for protecting the ecological environment and public health.

Intranasal Dexmedetomidine in Elderly Patients (Aged > 65 Years) During Maxillofacial Surgery: Sedative Properties and Safety Analysis.

PURPOSE: Light sedation rather than intravenous sedation is preferred when patients have a low heart rate and blood pressure during maxillofacial surgery. Intranasal administration of dexmedetomidine is reported to be efficacious and safe in adults. However, dexmedetomidine could be unsuitable for routine clinical use in elderly patients because many of these patients take ß-blockers, which increase the cardiovascular effects of dexmedetomidine. The objectives of the study were to evaluate the sedative properties and safety of intranasal dexmedetomidine, regardless of concurrent ß-blocker treatment, in elderly patients who underwent maxillofacial surgery. METHODS: This study was a retrospective analysis of 535 patients aged > 65 years (American Society of Anesthesiologists physical status I or II) who were undergoing maxillofacial surgery. Very anxious patients and those with hypertension received intranasal 1 µg/kg dexmedetomidine through an intranasal mucosal atomization device before anesthesia (local ropivacaine). RESULTS: Intranasal administration of dexmedetomidine decreased the requirement for midazolam before surgery (18 of 252 vs 63 of 283, P < .0001), but increased the requirement for norepinephrine (102 of 252 vs 8 of 283, P < .0001) during or after the surgery. A combination of a ß-blocker and intranasal administration of dexmedetomidine reduced the hemodynamic parameters for an extended period. Intranasal administration of dexmedetomidine resulted in bradycardia and hypotension, regardless of concurrent ß-blocker treatment. CONCLUSIONS: Intranasal 1 µg/kg dexmedetomidine was associated with a high sedation score during the operation, but also with bradycardia and hypotension.

Spinal Anesthesia With Peripheral Nerve Block Versus General Anesthesia With Peripheral Nerve Block for Elective Foot and Ankle Surgeries: A Retrospective Single-Center Study.

Peripheral nerve blocks are the regional techniques in orthopedic surgeries to control postoperative pain and have early discharge from hospital. However, anesthesia protocols for foot and ankle surgeries of institutes do not include multimodal analgesics including peripheral nerve blocks. The objective of the study was to compare spinal anesthesia with peripheral nerve block against general anesthesia with peripheral nerve block for elective foot and ankle surgeries. Patients have treated for elective foot and ankle surgery under general anesthesia (using propofol, 0.05 mg/kg dezocine, and 1% sevoflurane; GA cohort, n = 112) or spinal anesthesia (using 0.5% bupivacaine, propofol, and 0.05 mg/kg dezocine; SA cohort, n = 132) or patients have treated for elective for foot and ankle surgery under general anesthesia (GL cohort, n = 115) or spinal anesthesia (SL cohort, n = 160) with the use of peripheral nerve block (the sciatic nerve blocks and adductor canal nerve blocks using 0.25% bupivacaine and 0.1 mg/kg dexamethasone). Propofol was administered in fewer amounts if anesthesia was used with the peripheral nerve block. Patients of the GL cohort were transferred to ward 36 minutes (mean) earlier than those of the SL cohort. None of the patients of the GL and the SL cohorts have received intraoperative opioid(s) for the management of pain. Patients of the GL and the SL cohorts have reported postoperative falls within 1 day after surgeries during movement. Patients of the SL cohort experienced more frequently difficulty with sleeping. Patients of the GL and the GA cohorts have reported nausea and vomiting. Only patients of the GL cohort were required usage of vasoactive drugs. The study provides information to anesthesiologists and surgeons regarding anesthesia techniques for elective foot and ankle surgeries for better surgical outcomes (Technical Efficacy Stage: 4).

Efficacy and Safety of Docetaxel and Sodium Cantharidinate Combination vs. Either Agent Alone as Second-Line Treatment for Advanced/Metastatic NSCLC With Wild-Type or Unknown EGFR Status: An Open-Label, Randomized Controlled, Prospective, Multi-Center Phase III Trial (Cando-L1).

Second-line treatment options for advanced/metastatic non-small cell lung cancer (NSCLC) patients are limited. We aimed to evaluate the efficacy and safety of docetaxel/sodium cantharidinate combination vs. either agent alone as second-line treatment for advanced/metastatic NSCLC patients with wild-type or unknown EGFR status. A randomized, open-label, phase III study was performed at 12 institutions. Patients with failure of first-line platinum regimens were randomized to receive either single-agent sodium cantharivsdinate (SCA) or single-agent docetaxel (DOX) or docetaxel/sodium cantharidinate combination (CON). The primary endpoints were centrally confirmed progression-free survival (PFS) and overall survival (OS). The secondary endpoints were objective response rate (ORR), disease control rate (DCR), quality of life (QoL) and toxicity. A total of 148 patients were enrolled in our study between October 2016 and March 2020. After a median follow-up time of 8.02 months, no significant difference was observed among the three groups in ORR (SCA vs. DOX vs. CON: 6.00% vs. 8.33% vs. 10.00%, respectively; p=0.814) and DCR (74.00% vs. 52.00% vs. 62.50%, respectively; p=0.080). In additional, the mOS was significantly higher in the CON group, compared with the single-agent groups (7.27 vs. 5.03 vs. 9.83 months, respectively; p=0.035), while no significant differences were observed in terms of PFS (2.7 vs. 2.9 vs. 3.1 months, respectively; p=0.740). There was no significant difference in the baseline QoL scores between the three groups (p>0.05); after treatment, life quality in SCA and CON group was significantly better than that in the DOX group (p<0.05). Furthermore, the incidence of adverse events (AEs) in the SCA group was significantly lower (46.00 vs. 79.17 vs. 25.00%, respectively; p=0.038) and the incidence of grade ≥3 AEs was also significantly lower in the SCA group compared with the DOX and CON groups (10.00 vs. 82.00 vs. 30.00%, respectively; p=0.042). Single-agent SCA and single-agent DOX has similar therapeutic efficacy in the second-line treatment of advanced/metastatic NSCLC with wild-type or unknown EGFR status, but single-agent SCA has fewer AEs and better QoL. Also, SCA plus DOX can significantly improve OS and exerted a significant synergistic effect, with good safety and tolerance profile.

RORα Suppresses Epithelial-to-Mesenchymal Transition and Invasion in Human Gastric Cancer Cells via the Wnt/ß-Catenin Pathway.

Retinoid-related orphan receptor alpha (RORα) is involved in tumor development. However, the mechanisms underlying RORα inhibiting epithelial-to-mesenchymal transition (EMT) and invasion are poorly understood in gastric cancer (GC). This study revealed that the decreased expression of RORα is associated with GC development, progression, and prognosis. RORα suppressed cell proliferation, EMT, and invasion in GC cells through inhibition of the Wnt/ß-catenin pathway. RORα overexpression resulted in the decreased Wnt1 expression and the increased RORα interaction with ß-catenin, which could lead to the decreased intranuclear ß-catenin and p-ß-catenin levels, concomitant with downregulated T-cell factor-4 (TCF-4) expression and the promoter activity of c-Myc. The inhibition of Wnt/ß-catenin pathway was coupled with the reduced expression of Axin, c-Myc, and c-Jun. RORα downregulated vimentin and Snail and upregulated E-cadherin protein levels in vitro and in vivo. Inversely, knockdown of RORα attenuated its inhibitory effects on Wnt/ß-catenin pathway and its downstream gene expression, facilitating cell proliferation, EMT, migration, and invasion in GC cells. Therefore, RORα could play a crucial role in repressing GC cell proliferation, EMT, and invasion via downregulating Wnt/ß-catenin pathway.

Cepharanthine promotes the effect of dexmedetomidine on the deposition of ß-amyloid in the old age of the senile dementia rat model by regulating inflammasome expression.

AIM OF THE STUDY: The present study evaluates the synergistic effect of cepharanthine (CP) with dexmedetomidine (DEM) on the deposition of ß-amyloid (Aß) in the brain tissue of senile dementia (SD) rats. MATERIAL AND METHODS: Senile dementia was induced by injecting D-gal intraperitoneally 60 mg/kg/day for six weeks and Aß1-42 (5 µl) intracranially. The effect of cepharanthine and dexmedetomidine was estimated by determining the cognitive function and neurological function score. Moreover, mediators of inflammation, parameters of oxidative stress and reactive oxygen species (ROS) were determined in the brain tissue of senile dementia rats. Mitochondrial membrane permeability and deposition of Aß1-42 was estimated in senile dementia rats. Western blot assay and reverse transcription polymerase chain reaction (RT-PCR) was performed for the expression of proteins and genes in the brain tissue of senile dementia rats. RESULTS: Data of the study reveal that cepharanthine alone and in combination with dexmedetomidine improves the neurological function score and cognitive function in SD rats. Moreover, parameters of oxidative stress, inflammatory mediators and production of ROS in CP, DEM and CP + DEM treated groups were compared to the SD group of rats. Treatment with CP, DEM and CP + DEM ameliorates the altered expression of NLRP3 pathway and deposition of Aß in the brain tissue of SD rats. CONCLUSIONS: In conclusion, data reveal that cepharanthine ameliorates the deposition of Aß and NLRP3 pathway in SD rats. Moreover, cepharanthine treatment with dexmedetomidine shows the synergistic effect against the aged SD rat model.

Diallyl disulfide inhibits TGF­ß1­induced upregulation of Rac1 and ß­catenin in epithelial­mesenchymal transition and tumor growth of gastric cancer.

Transforming growth factor­ß1 (TGF­ß1) has been demonstrated to promote epithelial­mesenchymal transition (EMT), invasion and proliferation in tumors via the activation of Rac1 and ß­catenin signaling pathways. The present study investigated the effects of diallyl disulfide (DADS) on TGF­ß1­induced EMT, invasion and growth of gastric cancer cells. TGF­ß1 treatment augmented EMT and invasion, concomitantly with increased expression of TGF­ß1, Rac1 and ß­catenin in gastric cancer cells. DADS downregulated the expression levels of TGF­ß1, Rac1 and ß­catenin. DADS, TGF­ß1 receptor inhibitor as well as Rac1 inhibitor antagonized the upregulation of the TGF­ß1­induced expression of these genes, abolishing the enhanced effects of TGF­ß1 on EMT and invasion. Blocking the TGF­ß1 receptor through inhibition resulted in the decreased expression of Rac1 and ß­catenin. Rac1 inhibitor reduced the TGF­ß1­induced ß­catenin expression. In addition, DADS and the aforementioned inhibitors attenuated the TGF­ß1­induced tumor growth and the expression changes of E­cadherin, vimentin, Ki­67 and CD34 in nude mice. These data indicated that the blockage of TGF­ß1/Rac1 signaling by DADS may be responsible for the suppression of EMT, invasion and tumor growth in gastric cancer.

Identification of potential targets for differentiation in human leukemia cells induced by diallyl disulfide.

Diallyl disulfide (DADS) is a primary component of garlic, which has chemopreventive potential. We previously found that moderate doses (15-120 µM) of DADS induced apoptosis and G2/M phase cell cycle arrest. In this study, we observed the effect of low doses (8 µM) of DADS on human leukemia HL-60 cells. We found that DADS could inhibit proliferation, migration and invasion in HL-60 cells, and arrested cells at G0/G1 stage. Then, cell differentiation was displayed by morphologic observation, NBT reduction activity and CD11b evaluation of cytometric flow. It showed that DADS induced differentiation, reduced the ability of NBT and increased CD11b expression. Likewise, DADS inhibited xenograft tumor growth and induced differentiation in vivo. In order to make sure how DADS induced differentiation, we compared the protein expression profile of DADS-treated cells with that of untreated control. Using high resolution mass spectrometry, we identified 18 differentially expressed proteins after treatment with DADS, including four upregulated and 14 downregulated proteins. RT-PCR and western blot assay showed that DJ-1, cofilin 1, RhoGDP dissociation inhibitor 2 (RhoGDI2), Calreticulin (CTR) and PCNA were decreased by DADS. These data suggest that the effects of DADS on leukemia may be due to multiple targets for intervention.

Diallyl disulfide suppresses epithelial-mesenchymal transition, invasion and proliferation by downregulation of LIMK1 in gastric cancer.

Diallyl disulfide (DADS) has been shown to have multi-targeted antitumor activities. We have previously discovered that it has a repressive effect on LIM kinase-1 (LIMK1) expression in gastric cancer MGC803 cells. This suggests that DADS may inhibit epithelial-mesenchymal transition (EMT) by downregulating LIMK1, resulting in the inhibition of invasion and growth in gastric cancer. In this study, we reveal that LIMK1 expression is correlated with tumor differentiation, invasion depth, clinical stage, lymph node metastasis, and poor prognosis. DADS downregulated the Rac1-Pak1/Rock1-LIMK1 pathway in MGC803 cells, as shown by decreased p-LIMK1 and p-cofilin1 levels, and suppressed cell migration and invasion. Knockdown and overexpression experiments performed in vitro demonstrated that downregulating LIMK1 with DADS resulted in restrained EMT that was coupled with decreased matrix metalloproteinase-9 (MMP-9) and increased tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. In in vitro and in vivo experiments, the DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicate that downregulation of LIMK1 by DADS could explain the inhibition of EMT, invasion and proliferation in gastric cancer cells.

[A multicenter, randomized, controlled, phase â ¢ clinical study of PEG-rhG-CSF for preventing chemotherapy-induced neutropenia in patients with breast cancer and non-small cell lung cancer].

OBJECTIVE: To explore the safety and efficacy of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in preventing chemotherapy-induced neutropenia in patients with breast cancer and non-small cell lung cancer (NSCLC), and to provide the basis for clinical application. METHODS: According to the principle of open-label, randomized, parallel-group controlled clinical trial, all patients were randomized by 1∶1∶1 into three groups to receive PEG-rhG-CSF 100 µg/kg, PEG-rhG-CSF 6 mg, or rhG-CSF 5 µg/kg, respectively. The patients with breast cancer received two chemotherapy cycles, and the NSCLC patients received 1-2 cycles of chemotherapy according to their condition. All patients were treated with the combination chemotherapy of TAC (docetaxel+ epirubicin+ cyclophosphamide) or TA (docetaxel+ epirubicin), or the chemotherapy of docetaxel combined with carboplatin, with a 21 day cycle. RESULTS: The duration of grade 3-4 neutropenia in the PEG-rhG-CSF 100 µg/kg and PEG-rhG-CSF 6 mg groups were similar with that in the rhG-CSF 5 µg/kg group (P>0.05 for all). The incidence rate of grade 3-4 neutropenia in the PEG-rhG-CSF 100 µg/kg group, PEG-rhG-CSF 6 mg group, and G-CSF 5 µg/kg group were 69.7%, 68.4%, and 69.5%, respectively, with a non-significant difference among the three groups (P=0.963). The incidence rate of febrile neutropenia in the PEG-rhG-CSF 100 µg/kg group, PEG-rhG-CSF 6 mg group and G-CSF 5 µg/kg group were 6.1%, 6.4%, and 5.5%, respectively, showing no significant difference among them (P=0.935). The incidence rate of adverse events in the PEG-rhG-CSF 100 µg/kg group, PEG-rhG-CSF 6 mg group and G-CSF 5 µg / kg group were 6.7%, 4.1%, and 5.5%, respectively, showing a non-significant difference among them (P=0.581). CONCLUSIONS: In patients with breast cancer and non-small cell lung cancer (NSCLC) undergoing TAC/TA chemotherapy, a single 100 µg/kg injection or a single fixed 6 mg dose of PEG-rhG-CSF at 48 hours after chemotherapy show definite therapeutic effect with a low incidence of adverse events and mild adverse reactions. Compared with the continuous daily injection of rhG-CSF 5 µg/kg/d, a single 100 µg/kg injection or a single fixed 6 mg dose of PEG-rhG-CSF has similar effect and is more advantageous in preventing chemotherapy-induced neutropenia.

Identification of potential targets for diallyl disulfide in human gastric cancer MGC-803 cells using proteomics approaches.

Diallyl disulfide (DADS) is characterized as an effective agent for the prevention and therapy of cancer, however, mechanisms regarding its anticancer effects are not fully clarified. In the present study, we compared the protein expression profile of gastric cancer MGC-803 cells subjected to DADS treatment with that of untreated control cells to explore potential molecules regulated by DADS. Using proteomic approaches, we identified 23 proteins showing statistically significant differences in expression, including 9 upregulated and 14 downregulated proteins. RT-PCR and western blot analysis confirmed that retinoid-related orphan nuclear receptor α (RORα) and nM23 were increased by DADS, whereas LIM kinase-1 (LIMK1), urokinase-type plasminogen activator receptor (uPAR) and cyclin-dependent kinase-1 (CDK1) were decreased. DADS treatment and knockdown of uPAR caused suppression of ERK/Fra-1 pathway, downregulation of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9) and vimentin, and upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3) and E-cadherin, concomitant with inhibition of cell migration and invasion. Moreover, knockdown of uPAR potentiated the effects of DADS on MGC-803 cells. These data demonstrate that downregulation of uPAR may partially be responsible for DADS-induced inhibition of ERK/Fra-1 pathway, as well as cell migration and invasion. Thus, the discovery of DADS-induced differential expression proteins is conducive to reveal unknown mechanisms of DADS anti-gastric cancer.

Casticin inhibits self-renewal of liver cancer stem cells from the MHCC97 cell line.

Casticin exerts anticarcinogenic activity in several types of cancers, including human hepatocellular carcinoma (HCC). The aim of the present study was to investigate the effects of casticin, which is derived from Fructus Viticis Simplicifoliae, on the self-renewal capacity of liver cancer stem cells (LCSCs) derived from the HCC MHCC97 cell line. The present study demonstrated that casticin significantly inhibited the proliferation of LCSCs from the MHCC97 cell line in a dose-dependent manner (P<0.05), the half maximal inhibitory concentration of the parental cells and LCSCs was 17.9 and 0.5 µmol/l, respectively. Furthermore, casticin reduced the sphere-forming capacity of LCSCs and downregulated ß-catenin protein expression in a concentration-dependent manner. Lithium chloride, an agonist known to activate the Wnt/ß-catenin signaling pathway, attenuated the casticin-induced downregulation of ß-catenin protein expression and inhibited the self-renewal capacity. To the best of our knowledge, the present study is the first to demonstrate that casticin effectively eradicates LCSCs and ß-catenin was identified as the potential target. Thus, casticin may offer a novel therapeutic approach for the treatment of HCC.

Casticin inhibits epithelial-mesenchymal transition of liver cancer stem cells of the SMMC-7721 cell line through downregulating Twist.

The existence of cancer stem cells (CSCs) is central to the pathogenesis and therapeutic target of human hepatocellular carcinoma. The aim of this study was to investigate the effects of casticin on epithelial-mesenchymal transition (EMT) of liver cancer stem cells (LCSCs) derived from the SMMC-7721 cell line. Our results demonstrated that CD133+ sphere-forming cells (SFCs) sorted from the SMMC-7721 cell line not only possessed a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in Balb/c-nu mice, indicating that CD133+ SFCs possessed similar traits to LCSCs. Casticin increased the expression levels of E-cadherin and decreased those of N-cadherin in LCSCs. Treatment of LCSCs with casticin for 48 h also decreased the levels of the EMT-associated transcription factor, Twist. Overexpression of Twist attenuated the casticin-induced regulation of E-cadherin and N-cadherin protein expression, as well as the EMT capacity of LCSCs. In conclusion, CD133+ SFCs of the SMMC-7721 cell line may represent a subpopulation of LCSCs with the characteristics of EMT. Furthermore, casticin targeted LCSCs through the inhibition of EMT by downregulating Twist.

Chk1, but not Chk2, is responsible for G2/M phase arrest induced by diallyl disulfide in human gastric cancer BGC823 cells.

Diallyl disulfide (DADS) has been shown to cause G2/M phase cell cycle arrest in several human cancers. Here we demonstrate a mechanism by which DADS induces G2/M phase arrest in BGC823 human gastric cancer cells via Chk1. From cell cycle gene array results, we next confirmed that cyclin B1 expression was decreased by DADS, while the expression of p21, GADD45α and p53 were increased. Despite the lack of change in Chk1 gene expression in response to DADS according to the array analysis, intriguingly overexpression of Chk1, but not Chk2, exhibited increased accumulation in G2/M phase. Moreover, overexpression of Chk1 promoted the effect of DADS-induced G2/M arrest. Augmented phosphorylation of Chk1 by DADS was observed in Chk1-transfected cells, followed by downregulation of Cdc25C and cyclin B1 proteins. In contrast, phosphorylated Chk2 showed no obvious change in Chk2-transfected cells after DADS treatment. Furthermore, knockdown of Chk1 by siRNA partially abrogated DADS-induced downregulation of Cdc25C and cyclin B1 proteins and G2/M arrest. In contrast, knockdown of Chk2 did not show these effects. Therefore, these data indicate that DADS may specifically modulate Chk1 phosphorylation, and DADS-induced G2/M phase arrest in BGC823 cells could result in part from Chk1-mediated inhibition of the Cdc25C/cyclin B1 pathway.

miR-185 is an independent prognosis factor and suppresses tumor metastasis in gastric cancer.

miR-185 has been identified as an important factor in several cancers such as breast cancer, ovarial cancer, and prostate cancer. However, its effect and prognostic value in gastric cancer are still poorly known. In this study, we found that the expression levels of miR-185 were strongly downregulated in gastric cancer and associated with clinical stage and the presence of lymph node metastases. Moreover, miR-185 might independently predict OS and RFS in gastric cancer. We further found that upregulation of miR-185 inhibited the proliferation and metastasis of gastric cancer cells in vitro and in vivo. Taken together, our findings demonstrate that the miR-185 is important for gastric cancer initiation and progression and holds promise as a prognostic biomarker to predict survival and relapse in gastric cancer. It is also a potential therapeutic tool to improve clinical outcomes in the above disease.

Anti-proliferative and pro-apoptotic effect of carvacrol on human hepatocellular carcinoma cell line HepG-2.

Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.

Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma.

AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC). METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry. RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC(50) was 4.68, 7.65, 8.96, 11.65 and 64.82 micromol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC(50) of 4.59 micromol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% +/- 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% +/- 0.73%, 4.5% +/- 1.3% and 11.5% +/- 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% +/- 0.04%) in the control group (P < 0.01). The positive area rates (19.0% +/- 5.7%, 12.2% +/- 3.5% and 5.2% +/- 1.6% ) of PCNA in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly lower than that (29.5% +/- 9.4%) in the control group (P < 0.05). rVBMDMP at doses of 1, 3 and 10 mg/kg significantly reduced the tumor microvessel area levels (0.26% +/- 0.07%, 0.12% +/- 0.03% and 0.05% +/- 0.01% vs 0.45% +/- 0.15%) in HepG2 xenografts (P < 0.01), as assessed by CD31 staining. CONCLUSION: rVBMDMP has effective and unique anti-tumor properties, and is a promising candidate for the development of anti-tumor drugs.