Effect of siRNA targeting Ets2 gene on chemosensitization of human acute monocytic leukemic cell line SHI-1

Objective:This study was to observe the levels of Ets2 mRNA expression in leukemia patients and investigate the effect of small interfering RNA(siRNA)targeting Ets2 gene on sensibility of human acute monocytic leukemic cell line SHI-1 cells to etoposide(VP-16).Methods:Ets2 mRNA levels were determined by reverse transcription polymerase chain reaction(RT-PCR).After the transfection of Ets2 siRNA to SHI-1 cells by electroporation method,qRT-PCR was used to detect Ets2 gene expression in these cells;VP-16-induced apoptosis was investigated by Annexin V-FITC/PI.Results:Est2 mRNA was detectable in SHI-cells.The Est2 expression rate was respectively 10%in 5 healthy volunteers,60%in 5 acute lymphocytic leukemia(ALL)patients,73.68%in 19 acute nonlymphocytic leukemia(ANLL)patients and 100%in 4 chronic myeloid leukemia(CML)patients.The expression levels of Ets2 mRNA were significantly higher in leukemia patients compared with healthy volunteers.It also showed that siRNA targeting Ets2 gene resulted in substantial loss of Ets2 mRNA of SHI-1 cells compared to the control groups.Downregulation of Ets2 gene expression increased SHI-1 cells apoptosis and VP-16-induced apoptosis of SHI-1 cells.Conclusion:The high-level expression of Ets2 transcription factor in leukemia cells were connected with proliferation and anti-apoptosis of leukemia cells.SiRNA mediated Ets2 gene silencing induced cell apoptosis and enhanced in vitro sensitivity to chemotherapy(VP-16)of SHI-1 cells.It speculated the high-level expression of Ets2 may actually be an unfavorable determinant of chemotherapy sensitivity in leukemia.

COX-2 blocking therapy in cisplatin chemosensitization of ovarian cancer:An allicin-based nanomedicine approach

Recently,the utilization of nonsteroidal anti-inflammatory drugs(NSAIDs)to sensitize cisplatin(CDDP)has gained substantial traction in the treatment of ovarian cancer(OC).However,even widely employed NSAIDs such as celecoxib and naproxen carry an elevated risk of cardiovascular events,notably throm-bosis.Furthermore,the diminished sensitivity to CDDP therapy in OC is multifactorial,rendering the ap-plication of NSAIDs only partially effective due to their cyclooxygenase-2(COX-2)inhibiting mechanism.Hence,in this study,reactive oxygen species(ROS)-responsive composite nano-hydrangeas loaded with the Chinese medicine small molecule allicin and platinum(IV)prodrug(DTP@AP NPs)were prepared to achieve comprehensive chemosensitization.On one front,allicin achieved COX-2 blocking therapy,en-compassing the inhibition of proliferation,angiogenesis and endothelial mesenchymal transition(EMT),thereby mitigating the adverse impacts of CDDP chemotherapy.Simultaneously,synergistic chemosensi-tization was achieved from multifaceted mechanisms by decreasing CDDP inactivation,damaging mito-chondria and inhibiting DNA repair.In essence,these findings provided an optimized approach for syner-gizing CDDP with COX-2 inhibitors,offering a promising avenue for enhancing OC treatment outcomes.

Chemosensitizing Effect of Monk Fruit Extract on Human Bladder Cancer Cells

The outcomes of chemotherapy have been unsatisfactory with the palpable side effects. We hypothesized that natural products might help improve chemotherapy with few side effects. Recently, we came across the bioactive extracts of monk fruit (Siraitia grosvenori) with anticancer activity. We then investigated if these extracts might have chemosensitizing effect to improve the efficacy of drugs clinically used today. Four different drugs, cisplatin (CPL), carboplatin (CBL), mitomycin C (MMC), and gemcitabine (GEM), were used in this study. Human bladder cancer T24 cells were treated with each drug itself or drug combined with either LLE or MOG (two types of monk fruit extracts). Cell viability was determined to assess anticancer effect and also explored the anticancer mechanism of such combinations, focusing on the status of glycolysis, cell cycle, and chromatin structure. Cell viability test showed that all drugs had anticancer activity, reducing cell viability, but only CPL showed the enhanced anticancer effect when combined with LLE (not with MOG). The rest of three drugs had no such effects with LLE or MOG. The CPL/LLE combination was found to disrupt glycolysis, by inhibiting hexokinase activity, resulted in the decreased ATP synthesis. This combination also blocked the cell cycle progression, due to a G1 cell cycle arrest. Moreover, the two epigenetic regulators, DNA methyltransferase and histone deacetylase, were inactivated with the combination, indicating chromatin modifications. Ultimately, these treated cells were found to undergo apoptosis. In conclusion, anticancer activity of CPL can be significantly enhanced with LLE. This chemosensitizing effect is attributed to the glycolysis inhibition, a G1 cell cycle arrest, and chromatin modifications, ultimately leading to apoptosis. Thus, certain natural products such as LLE could be used as an adjuvant agent in current chemotherapy, improving the drug efficacy but minimizing side effects.

Chemosensitization role of endocrine hormones in cancer chemotherapy

The endocrine changes of cancer patients on initial confirmed diagnosis and during chemotherapy havebeen gradually realized and clarified, as well as the dependences of different tumors on corresponding hormones. The prevalent endocrinotherapy suppresses tumor progression through downregulating dependent hormone level or completely inhibitting its combination with receptors. Because of the different and even antagonistic mechanisms, endocrinotherapy should not be used with chemotherapy at the same time, or it will reduce the chemotherapeutic efficacy, which is a widely accepted principle in clinic.

Roscovitine synergizes with conventional chemo-therapeutic drugs to induce efficient apoptosis of human colorectal cancer cells

AIM: To examine the ability of cyclin-dependent kinase inhibitor (CDKI) roscovitine (Rosco) to enhance the antitumor effects of conventional chemotherapeutic agents acting by different mechanisms against human colorectal cancer. METHODS: Human colorectal cancer cells were treat-ed, individually and in combination, with Rosco, taxol, 5-Fluorouracil (5-FU), doxorubicine or vinblastine. The antiproliferative effects and the type of interaction of Rosco with tested chemotherapeutic drugs were de-termined. Cell cycle alterations were investigated by fluorescence-activated cell sorter FACS analysis. Apop-tosis was determined by DNA fragmentation assay. RESULTS: Rosco inhibited the proliferation of tumor cells in a time-and dose-dependent manner. The ef-ficacies of all tested chemotherapeutic drugs were markedly enhanced 3.0-8.42 × 103 and 130-5.28 × 103 fold in combination with 5 and 10 μg/mL Rosco, re-spectively. The combination of Rosco and chemothera-peutic drugs inhibited the growth of human colorectal cancer cells in an additive or synergistic fashion, and in a time and dose dependent manner. Rosco induced apoptosis and synergized with tested chemothera-peutic drugs to induce efficient apoptosis in human colorectal cancer cells. Sequential, inverted sequential and simultaneous treatment of cancer cells with combi-nations of chemotherapeutic drugs and Rosco arrested the growth of human colorectal cancer cells at various phases of the cell cycle as follows: Taxol/Rosco (G2/M-and S-phases), 5-FU/Rosco (S-phase), Dox/Rosco (S-phase) and Vinb/Rosco (G2/M-and S-phases). CONCLUSION: Since the eff icacy of many anticancer drugs depends on their ability to induce apoptotic cell death, modulation of this parameter by cell cycle inhibi-tors may provide a novel chemo-preventive and chemo-therapeutic strategy for human colorectal cancer.

Influence of Tamoxifen or the combination of Tamoxifen and Cisplatin on the growth of human lung adenocarcinoma A549 cells

The experiment aims to investigate the influence of Tamoxifen and the combination of Tamoxifen and Cisplatin （DDP） on the growth of human lung adenocarcinoma A549 cells. Methods： We treated human lung adenocarcinoma A549 cells with different concentrations of Tamoxifen, DDP and combination of DDP and Tamoxifen with non-toxicity for 72 h. Then we calculated the inhibition rate through MTT approach and detected the apoptosis rate by flow cytometry. The statistical analysis was performed with SPSS 13.0 software and statistical differences were determined by one-way ANOVA. The data were expressed as the mean ＋ standard deviation and all experiments were performed in three times. The value of P 〈 0.05 was considered to indicate a statistically significant difference. Results： 1. The inhibition rates of Tamoxifen with 2.5 pmol/L, 5 tJmol/L, 10 μmol/L, and 20 μmol/L on the growth of the A549 cells were 18.7%, 25.8%, 54% and 98.8%, respectively （P = 0.000）. Tamoxifen with concentration of 1 μmol/L has no obvious cytoxicity on the A549 cells （P 〉 0.05）. 2. As the increase concentration of Tarnoxifen, the S stage and G2/M of the A549 cells decreased while the G0/G1 increased. The apoptosis rate of Tamoxifen with 0 μmol/L, 0.1 μmol/L, 1 μmol/L and 10 μmol/L on the A549 cells were 6.51%, 8.91%, 17.97% and 42.7%, respectively. 3. The inhibition rates of combination of Tamoxifen with 1 μmol/L and DDP with 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL on the A549 cells were 40.4%, 54.4%, 72.9%, 86.1% and 92.4%, respectively （P 〈 0.05）. Conclusion： Tamoxifen can inhibit the proliferation of human lung adenocarcinoma A549 cells and induce the apoptosis of the A549 cells. The combination of Tamoxifen with non-toxicity and DDP can improve the sensitivity of chemotherapy on the A549 cells.

The Aryl Hydrocarbon Receptor: A Target for Breast Cancer Therapy

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a battery of genes in response to exposure to a broad class of environmental poly aromatic hydrocarbons (PAH). AhR is historically characterized for its role in mediating the toxicity and adaptive responses to these chemicals, however mounting evidence has established a role for it in ligand-independent physiological processes and pathological conditions, including cancer. The AhR is overexpressed and constitutively activated in advanced breast cancer cases and was shown to drive the progression of breast cancer. In this article we will review the current state of knowledge on the possible role of AhR in breast cancer and how it will be exploited in targeting AhR for breast cancer therapy.

Using ^(99m)Tc-MIBI to Evaluate the Effects of Chemosensitizer on P-glycoprotein in Multidrug-resistant Carcinoma Cells

To establish a method to evaluate the effects of chemosensitizer onP-glycoprotein using ^(99m)Tc-MIBI, and observe the changes of ^(99m)Tc-MIBI uptake kinetics andP-glycoprotein levels after using verapamil in MDR human breast cells MCF-7/Adr. Methods: MDR breastcarcinoma cells, MCF-7/Adr, were incubated and different protocols were performed. Protocol Ⅰ: achemosensitizer, verapamil (10 μmol/L), was added into cell culture medium, while in control group,the same volume of DMEM was given. Cells were harvested after 2 h incubation with ^(99m)Tc-MIBI.Protocol Ⅱ: Verapamil (10 μmol/L) was added into cell culture medium and incubated for 20 min, 40min, 60 min, 80 min, 8 h, 24 h, 48 h and 72 h respectively. Cells were harvested after 2 hincubation with ^(99m)Tc-MIBI. The radioactivity of the cells was measured and P-glycoproteinexpression levels were determined with immunohistochemical stain. Results: Protocol Ⅰ: After 2hincubation with verapamil the cellular uptake of ^(99m)Tc-MIBI was remarkably higher than controlgroup (t=2.33, P 【 0.05), but there was no difference in P-glycoprotein expression levels betweentwo groups (P 】 0.05). Protocol Ⅱ: In verapamil group, ^(99m)Tc-MIBI uptake was increased withincubation time prolonging (F=58.2, P 【 0.05). When verapamil incubation time surpassed 8 h the^(99m)Tc-MIBI uptake negatively correlated to the P-glycoprotein expression levels (r=-0.73, P 【0.01). However, when incubation time was less than 80 min, there was no correlation between^(99m)Tc-MIBI accumulation and P-glycoprotein levels (r=0.16, P 】 0.05). Conclusion: ^(99m)Tc-MIBImay be used to evaluate the qualitative as well as quantitative change of P-glycoprotein expressionlevels induced by the chemosensitizer, verapamil.

Downregulation of iASPP Expression Suppresses Proliferation, Invasion and Increases Chemosensitivity to Paclitaxel of Head and Neck Squamous Cell Carcinoma In Vitro

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma(HNSCC)and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC.This study investigated the function of iASPP playing in proliferation and invasion of HNSCC in vitro.Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group,respectively.The non-infected Tu686 cells were named the CON group.CCK-8 assay,flow cytometry,transwell invasion assay were performed to detect the effects of iASPP inhibition in vitro.Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h(F=32.459,P=0.000),96 h(F=51.407,P=0.000),120 h(F=35.125,P=0.000)post-transfection,was significantly lower than that of shRNANC cells and CON cells.The apoptosis ratio of shRNA-iASPP cells was 9.42%±0.39%(F=299.490,P=0.000),which was significantly higher than that of CON cells(2.80%±0.42%)and shRNA-NC cells(3.18%±0.28%).The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65%±1.09%(F=388.901,P=0.000),which was strikingly increased,compared with that of CON cells(55.19%±1.02%)and shRNA-NC cells(54.62%±0.88%).The number of invading cells was 56±4 in the shRNA-iASPP group(F=84.965,P=0.000),which decreased significantly,compared with the CON group(111±3)and the shRNA-NC group(105±8).The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased,compared with CON cells and shRNA-NC cells(F=634.841,P=0.000).Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.

Predictive value of MTT assay as an in vitro chemosensitivity testing for gastric cancer:One institution's experience

AIM:To investigate the predictive clinical value of in vitro 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay for directing chemosensitivity in patients with gastric cancer. METHODS:Results of a total of 353 consecutive patients with gastric cancer treated with MTT-directed chemotherapy or physician’s empirical chemotherapy from July 1997 to April 2003 were reviewed and analyzed retrospectively. RESULTS:The overall 5-year survival rate of MTT- sensitive group (MSG) and control group (CG) was 47.5% and 45.1%, respectively. The results of subgroup analysis with Cox proportional-hazards model were favorable for the MSG-sensitive group. However, no statistically significant difference in survival rate was observed between the two groups. CONCLUSION:Individualized chemotherapy based on in vitro MTT assay is beneficial, but needs to be confirmed by further randomized controlled trials.

Metabolomic studies of human gastric cancer:Review

Metabolomics is a field of study in systems biology that involves the identification and quantification of metabolites present in a biological system. Analyzing metabolic differences between unperturbed and perturbed networks, such as cancerous and noncancerous samples, can provide insight into underlying disease pathology, disease prognosis and diagnosis. Despite the large number of review articles concerning metabolomics and its application in cancer research, biomarker and drug discovery, these reviews do not focus on a specific type of cancer. Metabolomics may provide biomarkers useful for identification of early stage gastric cancer, potentially addressing an important clinical need. Here, we present a short review on metabolomics as a tool for biomarker discovery in human gastric cancer, with a primary focus on its use as a predictor of anticancer drug chemosensitivity, diagnosis, prognosis, and metastasis.

Role of microRNAs in gastric cancer

Although gastric cancer(GC)is one of the leading causes of cancer-related death,major therapeutic advances have not been made,and patients with GC still face poor outcomes.The prognosis of GC also remains poor because the molecular mechanisms of GC progression are incompletely understood.MicroRNAs(miRNAs)are noncoding RNAs that are associated with gastric carcinogenesis.Studies investigating the regulation of gene expression by miRNAs have made considerable progress in recent years,and abnormalities in miRNA expression have been shown to be associated with the occurrence and progression of GC.miRNAs contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors,affecting cell proliferation,apoptosis,motility,and invasion.Moreover,a number of miRNAs have been shown to be associated with tumor type,tumor stage,and patient survival and therefore may be developed as novel diagnostic or prognostic markers.In this review,we discuss the involvement of miRNAs in GC and the mechanisms through which they regulate gene expression and biological functions.Then,we review recent research on the involvement of miRNAs in GC prognosis,their potential use in chemotherapy,and their effects on Helicobacter pylori infections in GC.A greater understanding of the roles of miRNAs in gastric carcinogenesis could provide insights into the mechanisms of tumor development and could help to identify novel therapeutic targets.

Cyclin Dl antisense oligodexoyneucleotides inhibits growth and enhances chemosensitivity in gastric carcinoma cells

AIM： To examine the effects of cyclin D1 antisense oligodexoyneucleotides （ASODN） on growth and chemosensitivity of gastric carcinoma cell lines SGC7901 and its mechanism. METHODS： Phosphorothioate modified cyclin D1 ASODN was encapsulated by LipofectAMINE2000 （LF2000） and transfected into cells, the dose-effect curves and growth curves were observed. 5-FU, MTX, CDDP of different concentrations were given after transfecting cells with cyclin D1 ASODN for 24 h, the dose-effect responses were observed and IC50s were calculated. The mRNA expression of cyclin D1, thymidylate synthase （TS）, thymidine phosphorylase （TP） and dihydrofolate reductase （DHFR） was detected by reverse transcription-PCR （RTPCR） at 24 h and 48 h after transfection. RESULTS： Dose-dependent inhibitory effect was caused by cyclin D1 ASODN in SGC7901 cells. Transfecting gastric carcinoma cells with 0.2 μmol/L cyclin D1 ASODN for 24 h could inhibit growth significantly and reduce expression of cyclin D1 mRNA. Cyclin D1 ASODN could increase the chemosensitivity to 5-FU, MTX, CDDP in cells, The IC50s of different chemotherapeutic agents in ASODN plus chemotherapy groups were significantly lower than those in controls. Transfection with cyclin D1 ASODN leaded to an increase in TS and DHFR mRNA and a decrease in TP mRNA as determined by RT-PCR at 24 h, the alterations were more significant at 48 h. CONCLUSIONS： Cyclin D1 ASODN can decrease mRNA expression of cyclin D1, inhibit growth and enhance the chemosensitivity by changing the expression of enzymes related to metabolism of chemotherapeutic agents in SGC7901 gastric carcinoma cells.

Elucidation of the relationship of BNIP3 expression to gemcitabine chemosensitivity and prognosis

AIM: To evaluate the significance of BNIP3 in the pathogenesis of pancreatic cancer, we analyzed the relationship between the expression of BNIP3 and survival rate of the patients with pancreatic cancer, or chemosensitivities in pancreatic cancer cell lines, particularly for gemcitabine, the first-line anti-tumor drug for pancreatic cancer. METHODS: To compare the expression level of BNIP3 with the resistance to gemcitabine, eight pancreatic cancer cell lines were subjected to gemcitabine treatment and the quantitative real-time RT-PCR method was used to evaluate BNIP3 expression. Immunohistochemical analysis was also performed using 22 pancreatic cancer specimens to study relationship between BNIP3 expression and survival rate. RESULTS: Although no significantly positive association between BNIP3 mRNA level and gemcitabine chemosensitivity was observed, pancreatic cancer cell lines that were sensitive to gemcitabine treatment tended to show high levels of BNIP3 expression. The converse, an absence of BNIP3 expression, was not correlated with gemcitabine resistance. We further compared the BNIP3 expression profiles of resected primary pancreaticcancer specimens with the prognosis of the patients, and found a tendency of favorable prognosis and low BNIP3 expression. CONCLUSION: High levels of BNIP3 expression cannot be used as one of the predicting factors for gemcitabine chemosensitivity, and some yet to be known factors will have to fill the gap for the accurate prediction of pancreatic cancer chemosensitivity to gemcitabine. However, BNIP3 expression may have an impact on prediction of prognosis of patients with pancreatic cancer.

THE LOCAL AND GLOBAL EXISTENCE OF THE SOLUTIONS OF HYPERBOLIC-PARABOLIC SYSTEM MODELING BIOLOGICAL PHENOMENA

The authors prove the local existence and uniqueness of weak solution of a hyperbolic-parabolic system and establish the global existence of the weak solution for this system for the spatial dimension n = 1.

DIFFERENCE IN BIOLOGICAL CHARACTERISTICS AND SENSITIVITY TO CHEMOTHERAPY AND RADIOTHERAPY BETWEEN INTRAHEPATIC AND EXTRAHEPATIC CHOLANGIOCARCINOMA CELLS IN VITRO

Objective To investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro.Methods The intrahepatic and extrahepatic cholangiocarcinoma cell lines were established,and cells with steady passage were chosen to study the biological characteristics including morphology,growth dynamics,chromosome,and levels of cancer antigen(CA)125,CA19-9,alpha-fetoprotein(AFP),and carcino-embryonic antigen(CEA).Meanwhile,MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs,including cisplatin,paclitaxel,harringtonine,5-fluorouracil,vincristine,and aclacimomycin,and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured.Results The intrahepatic cholangiocarcinoma cells were mostly fusiform in shape,and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape.Their doubling time was 26.3 hours and 23.1 hours,respectively.Their average number of chromosomes was 59(range,38-84)and 67(range,49-103),respectively.The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid,while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells.The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously,while levels of other determined tumor markers in both kinds of cells were all within normal range.The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel,but not sensitive to the other 4 chemotherapeutic drugs.The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin,but not sensitive to the other 5 drugs.Both kinds of cells had poor sensitivity to radiotherapy.Conclusions Intrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape,doubling time,chromosome karyotype,tumor marker level,and chemosensitivity,whereas they both have poor radiosensitivity.Though they are similar in histopathology,they have different growth characteristics and have discrepancy in treatment and prognosis.

Low hypoxia inducible factor-1α(HIF-1α)expression in testicular germ cell tumors--a major reason for enhanced chemosensitivity?

The molecular basis for enhanced chemosensitivity of testicular germ cell tumors （GCT） has been an area of great interest, as it could potentially give us therapeutic leads in other resistant malignancies. Thus far, however, the increased sensitivity of C＆T has been variously attributed to multiple factors -- an inability to detoxify cisplatin, a lack of export pumps, an inability to repair the DNA damage, an intact apoptotic cascade and lack of p53 mutation; but a unifying underlying etiology leading to the aforementioned processes and having a translational implication has so far been elusive. Herein, we offer evidence to support a potential significant role for the previously demonstrated low hypoxia inducible factor-la （HIF-la） expression in mediating the general exquisite chemosensitivity of testicular GCT, through the aforementioned processes. This molecular mechanism based hypothesis could have a significant translational implication in platinum refractory GCT as well as other platinum resistant malignancies.

siRNA-mediated downregulation of TC21 sensitizes esophageal cancer cells to cisplatin

AIM： To determine the functional significance of TC21 in esophageal squamous cell carcinoma （ESCC）. METHODS： TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and tran- scripts were analyzed by reverse transcription-poly- merase chain reaction and protein by Western blotting.We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assess- ing the phosphorylation status of its downstream tar- gets, phosphoinositide 3-kinase （PI3K）, phosphatase and tensin homolog （PTEN）, protein kinase B （pAl〈t）, nuclear factor-KB （NF-~B） and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treat- ment was carried out by cell viability assay and cell cycle analysis using flow cytometry. RESULTS： TC21 knockdown in human ESCC cell line TEl3 cells, showed only a marginal increase （14.2%） in cell death compared with control cells. The expres- sions of the signaling proteins PI3K and pAkt, transcrip- tion factor NF-KB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregula- tion, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death （38.4%） was observed in cells treated with cis- platin after TC21 knockdown compared with cells which were treated with cisplatin alone （20% cell death）. CONCLUSION： Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-KB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo

Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Is there still a role for cytotoxic chemotherapy after targeted therapy and immunotherapy in metastatic melanoma? A case report and literature review

Metastatic melanoma has long been considered to have a very poor prognosis and to be chemo-resistant. However, a subgroup of patients with metastatic melanoma presents remarkable responses to chemotherapeutic agents, even in the absence of a response to modern targeted therapies and immunotherapies; accordingly, determining predictive biomarkers of the response to chemotherapies for metastatic melanoma remains a priority to guide treatment in these patients. We report a case study of a patient with B-Raf proto-oncogene serine/threonine kinase-mutated metastatic melanoma harbouring many genetic mutations. The patient did not respond to prior targeted therapies or immunotherapies but experienced a dramatic objective radiological and clinical response to subsequent dacarbazine-based chemotherapy. In the era of targeted therapies and immunotherapies for metastatic melanoma, cytotoxic chemotherapies may still represent an interesting therapeutic weapon in a well-deined subgroup of patients presenting with speciic genetic and molecular features.