Detection and comparison of tumor cell-associated microbiota from different compartments of colorectal cancer.

Introduction: Intratumoral microbes play an important role in the development of colorectal cancer (CRC). However, studying intratumoral microbes in CRC faces technical challenges, as tumor microbe communities are often contaminated by fecal microbes due to the structure of the gut folds and villi. The present study aimed to develop a new method for isolating tumor cell-associated microbiota and comparing microbial populations from different compartments. Materials and methods: The distribution of intestinal bacteria was detected using immunohistochemistry combined with 5R-16s rRNA gene sequencing to explore the effects of the sampling site and number of washes on the detection of microbiota. The 5R-16s rRNA gene sequencing was performed using 44 samples from 11 patients with CRC, including CRC tumor tissues (TT), normal tissues adjacent to CRC (NT), tumor cells (TC), and normal cells (NC). TC and NC were obtained from the TT and NT using an enzymatic digestion method. The microbiota and their potential functions in the four groups were analyzed and compared to determine the differential microbiota related to CRC. Results: Bacteria were mainly distributed in the feces covering intestinal tissues and in the epithelial cells and macrophages within the tissues. Different sampling sites and number of washes led to detection of different microbiota distributions. Although the cleaning method could be controlled, sampling sites varied and led to different microbiota distributions. The phyla of Firmicutes and Bacteroidetes were highly abundant in the conventionally used tissue samples, whereas Proteobacteria was the most abundant phyla in the cell samples isolated with the new method (i.e., after cell enzymatic hydrolysis). Detection of CRC cell-associated microbiota using a cell enzymatic digestion method showed that some bacteria, such as Fusobacterium, Eikenella, Shewanella, and Listeria, were more abundant in TT than NT, whereas the abundance of Akkermansia was lower in TT than NT. The tumor/normal ratios of some bacteria, such as Gemella, Escherichia, Shigella, and Blautia, were different between the cell and tissue samples. Conclusion: The cell enzymatic digestion method reduced fecal bacterial contamination, enabling low biomass intratumoral microbiota to be detected and allowing prediction of bacterial distributions.

KRT80 expression works as a biomarker and a target for differentiation in gastric cancer.

Keratin 80 (KRT80) is a filament protein that participates in cell differentiation and the integrity of the epithelial barrier. Here, KRT80 expression was higher in gastric cancer compared with normal mucosa at both mRNA and protein levels by bioinformatic analysis, qRT-PCR and Western blot (p<0.05), however, the methylation of KRT80 was lower than in normal mucosa (p<0.05). There was a negative relationship between promoter methylation and expression level of KRT80 gene in gastric cancer (p<0.05). KRT80 mRNA and protein expression was positively correlated with the differentiation of gastric cancer (p<0.05), while KRT80 methylation was negatively associated with gastric cancer differentiation and p53 mutation (p<0.05). The expression of KRT80 mRNA was positively linked to the short survival time of gastric cancers (p<0.05). The differential genes of KRT80 mRNA were involved in ligand-receptor interaction, estrogen signal pathway, peptidase, filament and cytoskeleton, keratinocyte differentiation, vitamin D receptor, muscle contraction, and B cell-mediated immunity (p<0.05). KRT80-related genes were classified into cell adhesion and junction, cadherin binding, skin and epidermis development, and so forth (p<0.05). KRT80 knockdown suppressed proliferation, anti-apoptosis, anti-pyroptosis, migration, invasion and epithelial-mesenchymal transition in gastric cancer cells (p<0.05). These findings indicated that up-regulated expression of KRT80 played a crucial part in gastric carcinogenesis, and might be considered as a biological marker for aggressive behaviors and poor prognosis. Its silencing might be used as an approach of target therapy for gastric cancer patients.

Short-term and long-term outcomes of patients with gastric cancer during versus before the COVID-19 pandemic: cohort study using propensity score matching method.

BACKGROUND: The negative effects of the novel coronavirus disease 2019 (COVID-19) pandemic on patients with gastric cancer are poorly understood. This study was designed to compare the short-term and long-term outcomes of patients with gastric cancer in the same period before and during the COVID-19 pandemic. METHODS: We retrospectively collected consecutive patients with definite diagnosis of gastric cancer at our center between 1 January and 30 June of 2019 (Before COVID-19) and 2020 (During COVID-19). A comparison was made between the number of patients and their characteristics before and during the COVID-19 epidemic. Propensity score matching (PSM) at 1:1 ratio was performed to evaluate the outcomes of patients that underwent laparoscopic radical gastrectomy in two groups. RESULT: The total number of patients diagnosed with gastric cancer during the COVID-19 pandemic increased by 21.4%, compared to that before the COVID-19 pandemic. AII the qualified patients were divided Before COVID-19 Pandemic group (BCP n = 99) and During COVID-19 Pandemic group (DCP n = 118). PSM yielded 81 patients with comparable baseline characteristics into each group. Compared to the BCP group, the DCP group had longer surgery time(P = 0.011), more blood loss(P = 0.015), longer postoperative hospital stay(P = 0.002). No statistical differences were observed in terms of type of resection, number of retrieved lymph nodes (LNs), pathology, short-term and long-term complications (P > 0.05). CONCLUSION: Patients diagnosed with gastric cancer during the COVID-19 pandemic had comparable short-term outcomes and long-term complications, but worse peri-operative outcomes, compared to that before the COVID-19 pandemic. Further research is needed to investigate long-term outcomes.

Bacteriological and oncological safety of complete laparoscopic radical resection of colorectal cancer without incision anastomosis based on propensity-matched scores.

Introduction: Complete laparoscopic radical resection of colorectal cancer without incision anastomosis is performed by means of natural orifice specimen extraction (NOSE), which avoids a large abdominal wall incision. Although this procedure is increasingly practiced, it is still underdeveloped, one reason being that controversy still exists regarding bacteriological and oncological safety. Aim: To demonstrate the safety of complete laparoscopic radical resection of colorectal cancer without incision anastomosis in bacteriology and oncology. Material and methods: This study was a retrospective study with prospectively collected data. This study continuously collected 420 patients who underwent colorectal cancer surgery in our hospital from January 2018 to March 2022. According to the different surgical methods, they were divided into the NOSE group (the natural orifice specimen extraction group) and the N-NOSE group (specimen removed through abdominal wall auxiliary incision). The two groups were matched 1 : 1 using the propensity score matching (PSM) method to balance at baseline. Bacteriological and oncological outcomes, short-term complications, and long-term prognosis were compared between the two groups. Results: One hundred and eighty-four of the 420 included patients were successfully matched. The differences in gender, body mass index, T stage, N stage, and diabetes status between the two groups after matching were not statistically significant. There were no significant differences in oncological outcomes, short-term complications, and 2-year postoperative disease-free survival between the two groups. There was no significant correlation between positive bacterial culture results and intra-abdominal infection. Conclusions: Compared with traditional laparoscopic surgery, complete laparoscopic radical resection of colorectal cancer without incision anastomosis has satisfactory bacteriological and oncological effects and is worthy of further clinical promotion.

Safety analysis of natural orifice specimen extraction surgery for colorectal cancer.

SUMMARY: This study investigated the safety, feasibility, and clinical outcomes of natural orifice specimen extraction surgery (NOSES) by collecting clinical from patients who underwent complete laparoscopic radical resection for colorectal cancer versus those who underwent conventional laparoscopic radical resection for colorectal cancer. Patients with colorectal cancer were selected as the study sample and grouped according to the different surgical methods. A total of 182 patients were eligible for enrollment in the study, including 92 patients who underwent NOSES (NOSES group) and 90 patients who underwent conventional laparoscopic radical colorectal cancer surgery. In the NOSES group, a total of 14 cases were observed to have a postoperative abdominal infection, and the remaining 78 cases did not have an abdominal infection, which we refer to as the infected and uninfected groups in this paper for further analysis. There was no difference in surgical outcome between NOSES surgery and conventional laparoscopic surgery. Diabetes mellitus, prolonged drain retention, and prolonged operative time were risk factors for the development of abdominal infection in NOSES. In contrast, intraoperative use of specimen retrieval bags, use of transanal endoscopic operations, and intraoperative flushing of the abdominal cavity with dilute iodophenol were protective factors for the development of postoperative abdominal infections. NOSES for colorectal cancer is worth promoting because of its small trauma and quick postoperative recovery.

A New Nomogram for Predicting the Postoperative Overall Survival in Patients with Middle-Aged and Elderly Rectal Cancer: A Single Center Retrospective Study in Chinese Population.

Purpose: Patients with middle-aged and elderly rectal cancer (MERC) usually have poor prognosis after surgery. This study aimed to develop a nomogram to achieve individualized prediction of overall survival (OS) in patients with MERC and to guide follow-up and subsequent diagnosis and treatment plans. Patients and Methods: A total of 349 patients were randomly assigned to the training and validation cohorts in a 7:3 ratio. Multivariate Cox regression analysis was performed using the results of univariate Cox regression analysis to confirm independent prognostic factors of OS. Thereafter, the nomogram was built using the "rms" package. Subsequently, discriminative ability and calibration of the nomogram were evaluated using receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). Integrated discrimination improvement (IDI), net reclassification improvement (NRI), and the area under the ROC curves (AUC) were compared between the nomogram and the tumor-node-metastasis (TNM) staging system (8th edition). Finally, we established a predictive model to assess the survival benefit of patients with MERC by calculating nomogram scores for each patient. Results: Six variables were identified as independent prognostic factors and included in the nomogram: smoking history, family history, hematochezia, tumor size, N stage, and M stage. Based on these factors, we successfully constructed a nomogram and evaluated its discriminative and predictive abilities using ROC curves, calibration curves, and DCA. ROC curves, IDI, and NRI showed that the nomogram had outstanding clinical utility compared with the TNM staging system (8th edition) for OS prediction. The predictive model successfully distinguished between high-, medium-, and low-risk MERC patients. Conclusion: Our nomogram provided a more satisfactory survival prediction ability than the TNM staging system (8th edition) for MERC patients. In addition, the nomogram was able to accurately categorize patients into different risk groups after surgery.

Transcriptional Regulation of ING5 and its Suppressive Effects on Gastric Cancer.

ING5 targets histone acetyltransferase or histone deacetylase complexes for local chromatin remodeling. Its transcriptional regulation and suppressive effects on gastric cancer remain elusive. Luciferase assay, EMSA, and ChIP were used to identify the cis-acting elements and trans-acting factors of the ING5 gene. We analyzed the effects of SAHA on the aggressive phenotypes of ING5 transfectants, and the effects of different ING5 mutants on aggressive phenotypes in SGC-7901 cells. Finally, we observed the effects of ING5 abrogation on gastric carcinogenesis. EMSA and ChIP showed that both SRF (-717 to -678 bp) and YY1 (-48 to 25bp) interacted with the promoter of ING5 and up-regulated ING5 expression in gastric cancer via SRF-YY1-ING5-p53 complex formation. ING5, SRF, and YY1 were overexpressed in gastric cancer, (P<0.05), and associated with worse prognosis of gastric cancer patients (P<0.05). ING5 had positive relationships with SRF and YY1 expression in gastric cancer (P<0.05). SAHA treatment caused early arrest at S phase in ING5 transfectants of SGC-7901 (P<0.05), and either 0.5 or 1.0 µM SAHA enhanced their migration and invasion (P<0.05). The wild-type and mutant ING5 transfectants showed lower viability and invasion than the control (P<0.05) with low CDC25, VEGF, and MMP-9 expression. Gastric spontaneous adenocarcinoma was observed in Atp4b-cre; ING5f/f, Pdx1-cre; ING5f/f, and K19-cre; ING5f/f mice. ING5 deletion increased the sensitivity of MNU-induced gastric carcinogenesis. ING5 mRNA might be a good marker of gastric carcinogenesis, and poor prognosis. ING5 expression was positively regulated by the interaction of SRF-YY1-ING5-p53 complex within the ING5 promoter from -50 bp upstream to the transcription start site. ING5 deletion might contribute to the tumorigenesis and histogenesis of gastric cancer.

Clinical effects of prophylactic transverse colostomy in patients undergoing completely laparoscopic transabdominal approach partial intersphincteric resection.

OBJECTIVE: To investigate the clinical effects of prophylactic transverse colostomy on gastrointestinal function recovery and complications in patients undergoing completely laparoscopic transabdominal approach partial intersphincteric resection (CLAPISR) of low rectal cancer. METHODS: We retrospectively analyzed the data of 74 patients with low rectal cancer who were treated with prophylactic transverse colostomy (Group A, n = 34) or without prophylactic transverse colostomy (Group B, n = 40). Surgery-related indicators, nutritional status indicators, systemic stress response indicators, and complications were compared between the two groups. RESULTS: On postoperative day 5, the C-reactive protein concentration and white blood cell count were not significantly different between the two groups; however, the serum concentrations of total protein and albumin were higher in Group A than in Group B. Within 26 months postoperatively, the total incidence rate of complications was not significantly different, but the incidence rate of anastomotic leakage was lower in Group A than in Group B. CONCLUSION: Prophylactic transverse colostomy based on CLAPISR can lead to faster recovery of gastrointestinal function, better improvement of postoperative nutritional indicators, and a lower incidence of anastomotic leakage. These characteristics are conducive to the rapid recovery of patients, making this procedure worthy of clinical application.

Analysis of the Clinical Effect of Natural Orifice Specimen Extraction Surgery Combined with Transanal Endoscopic Operations.

Background: To explore the clinical efficacy of natural orifice specimen extraction surgery (NOSES) combined with transanal endoscopic operations (TEOs) to remove specimens from the anus for laparoscopic radical resection of colorectal cancer. Methods: From January 2014 to May 2020, 120 colorectal cancer patients were selected as study samples, including 60 cases who underwent NOSES laparoscopic radical resection of colorectal cancer (experimental group) and 60 cases who underwent traditional laparoscopic radical resection of colorectal cancer (control group). The basic preoperative conditions, operative time, intraoperative blood loss, lymph node dissection, postoperative hospitalization time, postoperative exhaust and defecation time, postoperative anal function, and postoperative complications were analyzed retrospectively. Results: Sleep quality score (6.26 ± 1.16), pain score (2.95 ± 0.79), and hospitalization time (11.55 ± 3.79 days) were better than those for the control group (P < .05). There were no positive cases of incisal margin in both groups. Gastrointestinal function recovery time, blood loss, lymph node dissection, and postoperative complications were not statistically significant (P > .05). Conclusion: NOSES combined with TEOs has the advantages of less trauma, quick postoperative recovery, and low psychological pressure, so it is worth popularizing.

A Retrospective Study on the Feasibility of Completely Laparoscopic Transabdominal Approach Partial Intersphincteric Resection.

Background: With the development of surgical techniques, gastrointestinal surgeons seek ways to improve the quality of life (QOL) for postoperative patients, while ensuring the tumor radical effect, such as reducing trauma, alleviating pain, and preserving the anus. This article discusses the postoperative outcomes of completely laparoscopic transabdominal approach partial intersphincteric resection (CLTPISR). Materials and Methods: The clinical data of 68 patients who underwent CLTPISR at the Affiliated Hospital of Chengde Medical College between May 2017 and June 2020, including procedure-related data, general postoperative conditions, and postoperative anal function data, were retrospectively analyzed to investigate the feasibility and safety of CLTPISR. Results: All 68 rectal cancer patients completed the CLTPISR. The average age was (60.47 ± 7.08) years, the average operative time was (196.22 ± 8.15) minutes, the mean operative bleeding was (49.12 ± 29.61) mL, and the average postoperative hospital stay was (13.46 ± 4.78) days. The postoperative pathology showed that the circumferential margins and distal margins were all negative, the maximum tumor diameter was (3.26 ± 1.11) cm, the distance from the anal verge was (3.92 ± 0.89) cm, and the average number of lymph nodes cleared was (11.06 ± 3.67). There were 28 stage I patients (41.18%), 21 stage II patients (30.88%), and 19 stage III patients (27.94%). The median follow-up time after surgery was 28 months, and the rates of distant metastasis, disease-free survival, and overall survival of the patients were 8.82%, 91.18%, and 95.59%, respectively. The patient's postoperative anal function gradually recovered as time increased. Conclusions: Under the premise of strictly grasping the indications, the CLTPISR is a safe and feasible surgery. It could achieve maximum anal preservation and improve patients' QOL, while maintaining the tumor radical effect, and had the advantages of the short operation time, low intraoperative bleeding, and quick recovery. The Clinical Trial Registration number is LL2020397.

Construction, Validation, and Visualization of Two Web-Based Nomograms to Predict Overall and Cancer-Specific Survival in Patients with Gastric Cancer and Lung Metastases.

BACKGROUND: The lung is one of the most common sites of metastasis in gastric cancer. Our study developed two nomograms to achieve individualized prediction of overall survival (OS) and cancer-specific survival (CSS) in patients with gastric cancer and lung metastasis (GCLM) to better guide follow-up and planning of subsequent treatment. METHODS: We reviewed data of patients diagnosed with GCLM in the Surveillance, Epidemiology, and End Results (SEER) database from 2010 to 2015. The endpoints of the study were the OS and CSS. We used the "caret" package to randomly divide patients into training and validation cohorts in a 7 : 3 ratio. Multivariate Cox regression analysis was performed using univariate Cox regression analysis to confirm the independent prognostic factors. Afterward, we built the OS and CSS nomograms with the "rms" package. Subsequently, we evaluated the two nomograms through calibration curves, receiver operating characteristic (ROC) curves, and decision curve analysis (DCA). Finally, two web-based nomograms were built on the basis of effective nomograms. RESULTS: The OS analysis included 640 patients, and the results of the multivariate Cox regression analysis showed that grade, chemotherapy, and liver metastasis were independent prognostic factors for patients with GCLM. The CSS analysis included 524 patients, and the results of the multivariate Cox regression analysis showed that the independent prognostic factors for patients with GCLM were chemotherapy, liver metastasis, marital status, and tumor site. The ROC curves, calibration curves, and DCA revealed favorable predictive power in the OS and CSS nomograms. We created web-based nomograms for OS (https://zhenghh.shinyapps.io/aclmos/) and CSS (https://zhenghh.shinyapps.io/aslmcss/). CONCLUSIONS: We created two web-based nomograms to predict OS and CSS in patients with GCLM. Both web-based nomograms had satisfactory accuracy and clinical usefulness and may help clinicians make individualized treatment decisions for patients.

miR-496 inhibits proliferation via LYN and AKT pathway in gastric cancer.

MicroRNAs (miRNAs) operate as tumor suppressor or carcinogen to regulate cell proliferation, metastasis, invasion, differentiation, apoptosis, and metabolic process. In the present research, we investigated the effect and mechanism of miR-496 in human gastric cancer cells. miR-496 was downregulated in two gastric cancer cell lines, AGS and MKN45, compared with normal gastric epithelial cell line GES-1. miR-496 mimics inhibited the proliferation of AGS cells after the transfection for 48 and 72 h. The migration and invasion of AGS cells were also inhibited by the transfection of miR-496 mimics. miR-496 mimics induced the apoptosis through upregulating the levels of Bax and Active Caspase 3 and downregulating the levels of Bcl-2 and Total Caspase 3. Bioinformatics analysis showed that there was a binding site between miR-496 and Lyn kinase (LYN). miR-496 mimics could inhibit the expression of LYN in AGS cells. LYN overexpression blocked the inhibition of tumor cell growth, as well as the inhibition of AKT/mTOR signaling pathway induced by miR-496. In conclusion, miR-496 inhibited the proliferation through the AKT/mTOR signaling pathway via targeting LYN in gastric cancer cells. Our research provides a new potential target for clinical diagnosis and targeted treatment for gastric cancer.

Retrospective Study of Natural Orifice Specimen Extraction Surgery in Resection of Sigmoid and Rectal Tumors.

Background: With the development of surgical technology, surgeons are paying more and more attention to minimally invasive procedures such as injury reduction, pain reduction, and beautiful incisions to ensure the effectiveness of surgical treatment. This article discusses the safety, feasibility, and clinical outcomes of laparoscopic resection of sigmoid colon and rectal tumors via natural orifice specimen extraction surgery (NOSES). Materials and Methods: The clinical data of 39 patients who underwent complete laparoscopic resection of sigmoid colon tumor or rectal tumor at Chengde Medical College Hospital between 2018 and 2020, including general patient data (gender, age, body mass index [BMI], etc.), surgery-related data, general postoperative conditions, and postoperative pathological data, were retrospectively analyzed to explore the feasibility and safety of NOSES. Results: The specimens were all removed through the anorectal resection drag out type. The average age of 39 patients was 61.3 ± 10.2 years, the average BMI was 24.0 ± 3.1 kg/m2, the average postoperative hospital stay was 11.2 ± 4.4 days, 12 patients with sigmoid colon tumors, including 11 malignant tumors and 1 schwannoma, 27 rectal tumors, including 1 rectal villous tubular adenoma, among the 37 patients with malignant tumors, ulcer type 32 cases of adenocarcinoma and 5 cases of mass adenocarcinoma, mean number of lymph nodes detected intraoperatively (11.9 ± 3.9), mean operative time (162.9 ± 43.0 minutes), mean operative bleeding (36.9 ± 13.0 mL), mean time of initial exhaust (4.3 ± 3.0) days, mean time of laparoscopic drainage tube removal (9.8 ± 1.4) days, mean time of postoperative feeding (4.4 ± 3.0) days, the average maximum tumor diameter (3.7 ± 1.4 cm), and the average distance of the tumor from the anal margin (14.1 ± 6.1 cm); after surgery, there were two cases of anastomotic fistula. Conclusion: Laparoscopic resection of sigmoid colon and rectal tumors via natural orifice specimen extraction has the advantages of less pain, reduced incisional complications, good safety, and accurate efficacy in clinical applications.

The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer.

Dkk3 has been discovered during comparison of immortalized and parental cells. Its expression has been shown to reduce colony formation and induce apoptosis of cancer cells, acting as a tumor suppressor. Herein, we demonstrate that Dkk3 overexpression or protein treatment may inhibit colorectal cancer cell proliferation, migration, and invasion and that they may promote apoptosis and G2 phase arrest with hypoexpression of Bcl-2, cdc25B, cdc25c, N-cadherin, slug, and twist and hyperexpression of Bax and E-cadherin. This effect is consistent with that of recombinant Dkk3 exposure and blocked with anti-Dkk3 antibody. Dkk3 deletion in intestinal cells was not associated with the emergence of epithelial lesions; however, adenoma emerged after sodium desoxycholate treatment. At both mRNA and protein levels, Dkk3 expression was higher in normal than in cancer tissues (p<0.05). Dkk3 mRNA expression was negatively associated with its promoter methylation, growth pattern, differentiation, and favorable prognosis in the patients with colorectal cancer (p<0.05). Dkk3-related signal pathways in colorectal cancer included those of cellular adhesion and migration, melanogenesis, chemokine, Hedgehog, JAK-STAT, TOLL-like receptor, TGF-ß, MAPK, and calcium signaling (p<0.05). These findings indicate that Dkk3 expression levels can help assess cancer aggressiveness and patient prognosis. It might also suppress aggressive phenotypes and tumorigenesis as a molecular target in gene therapy.

The Roles of Beclin 1 Expression in Gastric Cancer: A Marker for Carcinogenesis, Aggressive Behaviors and Favorable Prognosis, and a Target of Gene Therapy.

Beclin 1 is encoded by Becn1, and plays a role in tumorigenesis, neurodegeneration, apoptosis and autophagy. Here, the aggressive phenotypes and relevant proteins were examined after Beclin 1 expression was altered in gastric cancer cells. We also observed the effects of Beclin 1 on gastric carcinogenesis using Becn1 knockout mice. Finally, clinicopathological significances of Beclin 1 expression were analyzed using meta- and bioinformatics analyses. Becn1 overexpression was found to inhibit proliferation, glucose metabolism, migration and invasion of gastric cancer cells, whereas its knockdown caused the opposite effects. Beclin 1 suppressed the tumor growth by decreasing proliferation and increasing apoptosis. The heterozygous abrogation of Becn1 in gastric pit, parietal and chief cells could not cause any epithelial lesion. Beclin 1-mediated chemoresistance was closely linked to the autophagy, Bax underexpression, and the overexpression of Bcl-2, LRP1, MDR1, and ING5. Bioinformatics analysis showed higher Becn1 mRNA expression in intestinal- than diffuse-type carcinomas (P<0.05), and in male than female gastric cancer patients (P<0.05). Becn1 hyperexpression was positively associated with both overall and progression-free survival rates of the cancer patients (P<0.05). Meta-analysis showed that down-regulated Beclin 1 expression in gastric cancer was positively with lymph node metastasis, TNM staging, dedifferentiation and poor prognosis (P<0.05). Becn1-related signal pathways in gastric cancer included prostate, lung, renal, colorectal, endometrial and thyroid cancers, glioma, and leukemia, the metabolism of amino acid, lipid and sugar, and some signal pathways of insulin, MAPK, TRL, VEGF, JAK-STAT, chemokine, p53, lysosome, peroxidome and ubiquitin-mediated protein degradation (P<0.05). These suggested that Beclin 1 might be considered as a potential marker of gastric carcinogenesis, aggressiveness and prognostic prediction, and as a target of gene therapy in gastric cancer.

Effect of Hsp90 Inhibitor KW-2478 on HepG2 Cells.

OBJECTIVE: The objectives of this study were to investigate the effects of proliferation, apoptosis, cell cycle, invasion, and senescence of KW-2478 on HepG2 cells, and to explore the related mechanism of apoptosis and the cell cycle. METHODS: HepG2 cells (hepatocellular carcinoma cells) were cultured with KW-2478, at different doses and for different times, in vitro. The MTT assay was used to detect the effect of KW-2478 on proliferation of HepG2 cells. Flow cytometry was used to determine the effects of KW-2478 on the cell cycle and apoptosis of HepG2 cells. The Transwell assay was used to determine the effect of KW-2478 on cell invasion. The ß-galactosidase assay tested the effect of low-dose KW-2478 on the senescence of HepG2 cells. Western blotting and the quantitative polymerase chain reaction were used respectively to assess changes in protein and mRNA levels of related factors in HepG2 cells after the KW-2478 treatment. RESULTS: KW-2478 significantly inhibited proliferation of HepG2 cells. KW-2478 induced apoptosis and cell cycle arrest of HepG2 cells, and inhibited the invasion of HepG2 cells; low dose KW-2478 promoted HepG2 senescence. CONCLUSION: KW-2478 inhibited the proliferation of HepG2 cells, induced apoptosis and cell cycle arrest, inhibited invasion, and promoted senescence. KW-2478 affected the expression of related factors in the mitochondrial apoptotic signaling and cell cycle-related regulatory pathways. KW-2478 downregulated the expression of STAT3, which is a key factor in the JAK-STAT pathway, indicating that KW-2478 may affect the function of HepG2 cells by downregulating STAT3.

Synthesis, in vitro stability, and antiproliferative effect of d-cysteine modified GnRH-doxorubicin conjugates.

Overexpression of gonadotropin-releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH, [d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH, and [d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH were conjugated with doxorubicin (Dox), respectively, through N-succinimidyl-3-maleimidopropionate as a linker to afford three new GnRH-Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP-HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor-positive MCF-7 human breast cancer cell line by MTT assay. The three GnRH-Dox conjugates showed improved metabolic stability in human serum in comparison with AN-152. The antiproliferative effect of conjugate II ([d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH-Dox) on MCF-7 cells was higher than that of conjugate I ([d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH-Dox) and conjugate III ([d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH-Dox), and the cytotoxicity of conjugate II against GnRH receptor-negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.

Effects of 17-allylamino-17-demethoxygeldanamycin on the induction of apoptosis and cell cycle arrest in HCT-116 cells.

The present study investigated the effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on apoptosis and the cell cycle of the HCT-116 human colon carcinoma cell line, with the aim of elucidating their underlying mechanisms. MTT was used to examine the inhibitory effects of 17-AAG on the proliferation of HCT-116 cells at various time points and doses. The cells were stained with Annexin V-fluorescein isothiocyanate/propidium iodide and evaluated by flow cytometry. The expression of signal transducer and activator of transcription (STAT)3, cyclin D1, cytochrome c (cyt-c), caspase 9 and caspase 3 at the mRNA and protein level was determined using reverse transcription-polymerase chain reaction and western blotting. Treatment with 17-AAG at a concentration of 1.25-20 mg/l for 24 and 48 h significantly inhibited the proliferation of HCT-116 cells in a time-dependent and concentration-dependent manner. Treatment with 17-AAG at concentrations of 1.25, 2.5 and 5 mg/l for 48 h significantly induced apoptosis and cell cycle arrest in HCT-116 cells. Exposure to 17-AAG at concentrations of 1.25, 2.5 and 5 mg/l for 48 h significantly downregulated the mRNA and protein expression of STAT3 and cyclin D1, but upregulated cyt-c, caspase 9 and caspase 3 in a concentration-dependent manner in HCT-116 cells. Therefore 17-AAG is able to inhibit cell proliferation, inducing apoptosis and G1 stage cell cycle arrest by downregulating the expression of cyclin D1, and promoting the mitochondria apoptosis by downregulating STAT3 in HCT-116 cells.

Effects of 17-AAG on the cell cycle and apoptosis of H446 cells and the associated mechanisms.

As a heat shock protein 90 inhibitor, 17-allylamino-17­demethoxygeldanamycin (17-AAG) has been studied in numerous types of cancer, however the effects of 17-AAG on apoptosis and the cell cycle of H446 cells remain unclear. In the current study, the MTT method was used to evaluate the inhibitory effects of different durations and doses of 17­AAG treatment on the proliferation of H446 cells. The cells were stained with Annexin-fluorescein isothiocyanate/propidium iodide and measured by flow cytometry, and the gene and protein expression levels of signal transducer and activator of transcription 3 (STAT3), survivin, cyclin D1, cyt­C, caspase 9 and caspase 3 were determined by reverse transcription­quantitative polymerase chain reaction and western blot analysis. The results indicated that with treatment with 1.25­20 mg/l 17­AAG for 24 and 48 h, significant inhibition of H446 cell proliferation was observed in a time­ and dose­dependent manner. With treatment of 3.125, 6.25 and 12.5 mg/l 17­AAG for 48 h, significant apoptosis and cell cycle arrest was observed. The results indicated that the gene and protein expression levels of STAT3, survivin and cyclin D1 were downregulated, and cyt­C, caspase 9 and caspase 3 were upregulated by 17­AAG in a dose-dependent manner when the cells were treated with 3.125 and 6.25 mg/l 17-AAG for 48 h. The results indicated that 17­AAG is able to inhibit the cell proliferation, induce apoptosis and G2/M arrest and downregulate the gene and protein expression levels of STAT3, survivin and cyclin D1, and upregulate gene and protein expression of cyt­C, caspase 9, caspase 3.

Paxillin expression is closely linked to the pathogenesis, progression and prognosis of gastric carcinomas.

Paxillin encodes a focal adhesion-associated protein and is involved in the progression and aggressive phenotypes of malignancies through its interactions with the actin cytoskeleton and key signal transduction oncogenes. The present study aimed to investigate the clinicopathological and prognostic significance of paxillin in gastric cancer. The expression of paxillin was evaluated using tissue microarrays of gastric adjacent non-cancerous mucosa, adenoma and carcinoma specimens by immunohistochemistry. Paxillin expression was compared against clinicopathological parameters and the survival time of the patients. Paxillin was highly expressed in gastric adenoma compared with that in non-neoplastic mucosa and carcinoma (P<0.05). Paxillin expression was lower in the younger carcinoma patients compared with that in the elder carcinoma patients (P<0.05). Paxillin expression was negatively correlated with tumor size, depth of invasion and lymph node metastasis, but not with patient gender, lymphatic or venous invasion, or TNM staging (P>0.05). Higher paxillin expression was observed in intestinal-type compared with diffuse-type carcinoma (P<0.05). Kaplan-Meier analysis indicated a positive association between paxillin expression and cumulative survival rate in all, advanced and intestinal-type carcinoma patients (P<0.05). Multivariate analysis using the Cox proportional hazards model indicated that patient age, depth of invasion, lymphatic invasion, lymph node metastasis, TNM staging and Lauren classification were independent prognostic factors for all gastric carcinomas (P<0.05). Aberrant paxillin expression may be involved in the growth, invasion, metastasis and differentiation of gastric carcinoma. Altered paxillin expression may, therefore, be employed as an indicator of pathobiological behaviors and prognosis of gastric carcinomas.