MiR-495 Inhibits Cisplatin Resistance and Angiogenesis in Esophageal Cancer by Targeting ATP7A.

Background: Cancer resistance to chemotherapy is closely associated with changes in transporter systems. In this study, we investigated the possible regulation of 1 copper ion transporter (ATP7A; ATPase copper transporting alpha) by microRNA miR-495 and its implications in cisplatin resistance and angiogenesis in esophageal cancer. Methods: MiR-495 and ATP7A mRNA expression in clinical tissue samples and 2 cancer cell lines (Eca-109 and TE1) were detected by quantitative real-time polymerase chain reaction. The levels of miR-495 and ATP7A expression in Eca-109 and TE1 cells were increased by transfection with miR-495 mimics and ATP7A-overexpression vectors. Cell proliferation, apoptosis, and angiogenesis were assessed by CCK-8, flow cytometry, and tube formation assays, respectively. The levels of TNF-α and VEGF in cell culture supernatants were detected by enzyme linked immunosorbent assay, and in situ expression of NLRP3 was measured by immunofluorescence. The binding of miR-495 to ATP7A sequences was verified by dual luciferase reporter assays. Results:ATP7A expression was significantly increased, while miR-495 expression was decreased in the cancer tissues of esophageal cancer patients. MiR-495 mimics decreased the proliferation and promoted the apoptosis of cisplatin-resistant Eca-109 and TE1 cells. Furthermore, tube formation by human umbilical vein endothelial cells, TNF-α and VEGF secretion, and the levels of MRP1, ABCG1, ABCA1, and NLRP3 expression in cisplatin-resistant Eca-109 and TE1 cells were all reduced by miR-495 mimics. MiR-495 was shown to directly bind to ATP7A gene sequences to repress ATP7A expression in Eca-109 and TE1 cells. ATP7A overexpression substantially abrogated the changes in proliferation, apoptosis, angiogenesis, and above-mentioned gene expression in cisplatin-resistant Eca-109 and TE1 cells. Conclusions: MiR-495 suppressed cisplatin resistance and angiogenesis in esophageal cancer cells by targeting ATP7A gene expression.

Icotinib is as efficacious as gefitinib for brain metastasis of EGFR mutated non-small-cell lung cancer.

BACKGROUND: The prognosis of non-small-cell lung cancer (NSCLC) with brain metastases is very poor. Currently, therapeutic methods for this patient population include whole-brain radiation therapy (WBRT), surgery, radiosurgery and systemic treatment. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) could be effective on cerebral metastases of mutated NSCLC. However, which EGFR-TKIs is more appropriate is still unknown. METHODS: We conducted a retrospective analysis of advanced NSCLC patients with brain metastases for EGFR targeted therapy from November 2013 to April 2018 at Dongguan People's Hospital, Southern Medical University, China. A total of 43 patients were recruit in this study. Among them, 21 cases received icotinib (125 mg, thrice a day) and 22 cases received gefitinib (250 mg, once a day) until disease progression or unacceptable toxicity. The primary end point of this study was intracranial PFS (iPFS). The relationships between therapeutic arms and patients characteristics were performed using Pearson's chi-square test or Fisher's exact test. The differences in PFS among the two arms were analyzed using Kaplan-Meier curves and log rank tests. RESULTS: There was no significant difference of intracranial ORR (66.6% versus 59.1%, P = 0.62) and DCR (85.7% versus 81.8%, P = 0.73) between the two arms. The median intracranial PFS (iPFS) for icotinib and gefitinib arms were 8.4 months (95% CI, 5.4 to 11.3 months) and 10.6 months (95% CI, 6.3 to 14.8 months), respectively (P = 0.17). Adverse events of the two study arms were generally mild. None of the patients experienced dose reduction of EGFR-TKIs. CONCLUSIONS: Our study showed that icotinib and gefitinib had similar efficacy for brain metastasis of EGFR mutated NSCLC. Large randomized studies are suggested to further illuminate the effect of these two EGFR-TKIs on cerebral lesions of NSCLC.

Expression of ATP7A in esophageal squamous cell carcinoma (ESCC) and its clinical significance.

OBJECTIVE: This study investigated the expression of P-type copper transporting adenosine triphosphatase ATP7A in the tumor tissues of patients with advanced esophageal squamous cell carcinoma (ESCC), and analyzed its correlation to clinicopathologic features and prognosis of advanced ESCC patients. METHODS: The expression of ATP7A protein in 49 specimens of advanced ESCC patients who were treated with first line cisplatin-based chemotherapy without surgery or radiotherapy, was detected by immunohistochemistry. The correlation of ATP7A expression with clinicopathologic features and prognosis of advanced ESCC patients weas analyzed by SPSS 16.0 statistical software package. RESULTS: Positive ATP7A staining was observed in cytoplasm of ESCC cells in 44. of tumors (22 of 49 cases), but was not detected in adjacent stroma of tumor tissue. ATP7A expression status was correlated with response to histologic grade and cisplatin-based chemotherapy (P values 0.02, 0.028 respectively). No significant association was found between ATP7A expression and age (P=0.085), gender (P=0.74), or PS (P=0.56). Kaplan-Meier analysis indicated that advanced ESCC patients positive for ATP7A positive had overall survival (OS) inferior to advanced ESCC patients who were ATP7A negative (P value was 0.037 by log-rank test). In univariate analysis, histologic grade and ATP7A expression were significantly correlated with OS (P=0.011 and 0.049 respectively); in multivariate analysis, histologic grade and ATP7A were independent factors significantly related to OS for advanced ESCC patients treated by cisplatin-based chemotherapy (P values 0.039 and 0.043 respectively). CONCLUSION: ATP7A was positively expressed in the majority of advanced ESCC tissues. The expression level of ATP7A was an important factor affecting tumor tissue's histologic grade, the response to platinum-based chemotherapy and the prognosis of advanced ESCC patients. This indicates that ATP7A might be involved in the genesis and development of ESCC, and could be a resistance marker for platinum-based chemotherapy, and a prognostic factor for survival in patients with ESCC treated by Pt-based chemotherapy.

The role of copper transporter ATP7A in platinum-resistance of esophageal squamous cell cancer (ESCC).

Purpose: Platinum derivatives, such as cisplatin (DDP), carboplatin and oxaliplatin, are widely used components of modern cancer chemotherapy including esophageal squamous cell cancer (ESCC). However, their roles are limited by the impact of intrinsic/acquired resistance mechanisms on tumor responses. Recent studies have shown that the mammalian copper transporters CTR1, ATP7A and ATP7B are involved in cisplatin-resistance to some cancers. Methods: The cytotoxicities of DDP in different cell lines were determined using the MTT assay. To determine whether knockdown the expression of ATP7A could reverse the platinum-resistance of EC109/DDP cells or not, we used RNA interference system to explore the role of ATP7A in platinum resistance. Results: We found that DDP-resistant cell sublines EC109/DDP (8.490 folds) showed cross-resistance to carboplatin (5.27 folds) and oxaliplatin (4.12 folds). ATP7A expressions in DDP-resistant cell sublines (EC109/DDP) were much higher than DDP-sensitive cell lines (EC109) at both mRNA and protein levels. ATP7A targeted small interfering RNA duplex at 100nM final concentration added into DDP-resistant cancer cells (EC109/DDP) markedly inhibited the expression of ATP7A as determined by Western blot (83.0%) and partially reversed DDP-resistance (37.09%), moreover, it also increased cell apoptosis at different DDP concentrations. Conclusions: These findings indicate that ATP7A high expression plays an important role in platinum-resistance of ESCC. This study sheds light on platinum resistance in ESCC patients and may have implications for therapeutic reversal of drug resistance.

[A novel assay for analysis of adenovirusmediated endosome lysis of T lymphocytes].

Objective: To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran (pH-sensitive fluorescent dye). Methods: After incubating Jurkat cells (T cell leukemia) with serotype 5 adenovirus (Ad5) and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy. Results: After incubating Jurkat cells with Ad5 and 80 µg/mL pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment. Conclusion: The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

Staged Improvement in Awareness of Disease for Elderly Cancer Patients in Southern China.

BACKGROUND: In mainland China, awareness of disease of elderly cancer patients largely relies on the patients' families. We developed a staged procedure to improve their awareness of disease. MATERIALS AND METHODS: Participants were 224 elderly cancer patients from 9 leading hospitals across Southern China. A questionnaire was given to the oncologists in charge of each patient to evaluate the interaction between family and patients, patient awareness of their disease and participation in medical decision-making. After first cycles of treatment, increased information of disease was given to patients with cooperation of the family. Then patient awareness of their disease and participation in medical decision-making was documented. RESULTS: Among the 224 cancer elderly patients, 26 (11.6%) made decisions by themselves and 125 (55.8%) delegated their rights of decision- making to their family. Subordinate family members tended to play a passive role in decision-making significantly. Patients participating more in medical decision-making tended to know more about their disease. However, in contrast to the awareness of disease, patient awareness of violation of medical recommendations was reversely associated with their participation in medical decision-making. Improvement in awareness of diagnosis, stages and prognosis was achieved in about 20% elderly cancer patients. About 5% participated more actively in medical decision-making. CONCLUSIONS: Chinese elderly cancer patient awareness of disease and participation in medical decision-making is limited and relies on their family status. The staged procedure we developed to improve patient awareness of disease proved effective.

Copper-transporting P-type adenosine triphosphatase (ATP7A) is associated with platinum-resistance in non-small cell lung cancer (NSCLC).

BACKGROUND: Copper export protein ATP7A is important for maintaining copper homeostasis. Recent studies have shown that copper transporters are also involved in the transport of platinum. The goal of this study was to determine the role of ATP7A in the platinum-resistance of non-small cell lung cancer (NSCLC). METHODS: Sensitivities to platinums were detected by MTT assay and drug-resistance related genes were analyzed by real-time PCR and immunoblotting between DDP-sensitive A549 and the corresponding DDP-resistant cell subline (A549/DDP). ATP7A expression was evaluated by immunohistochemistry in tumor tissues of unresectable NSCLC patients who received cisplatin-basing chemotherapy. RESULTS: The expression of ATP7A was significantly higher in A549/DDP cell subline than in A549 cells at both mRNA and protein levels. The silencing of ATP7A expression in A549/DDP by siRNA partially reversed DDP-resistance (29.62%) and increased cell apoptosis. ATP7A expression was detected in 41.6%of NSCLC patients, but not in adjacent stroma nor normal lung tissues. ATP7A-positive patients had a significantly poorer histological grade (p = 0.039) and poorer response to platinum-basing chemotherapy (p = 0.001) compared with ATP7A-negative patients. Cox's proportional hazards analysis showed that ATP7A expression was an independent prognostic factor for overall survival (p = 0.045). CONCLUSIONS: ATP7A overexpression played an important role in platinum-resistance of NSCLC, and was a negative prognostic factor of NSCLC patients treated with platinum-based chemotherapy.

Impact of KRAS mutation and PTEN expression on cetuximab-treated colorectal cancer.

AIM: To investigate the prognostic value of KRAS mutation, and phosphatase and tensin (PTEN) expression in Chinese metastatic colorectal cancer metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Ninety Chinese mCRC patients treated with cetuximab were evaluated for KRAS mutation and PTEN protein expression by DNA sequencing of codons 12 and 13 and immunohistochemistry, respectively. We then selected 61 patients treated with cetuximab, either in combination with chemotherapy, or alone as a second-line or third-line regimen to assess whether KRAS mutation or PTEN protein expression is associated with the response and the survival time of mCRC patients treated with cetuximab. RESULTS: KRAS mutation was found in 30 (33.3%) tumor samples from the 90 patients, and positive PTEN expression was detected in 58 (64.4%) of the 90 patients. Among the 61 patients who were treated with cetuximab as a second-line or third-line regimen, the resistance to cetuximab was found in 22 patients with KRAS mutation and in 39 patients without KRAS mutation, with a response rate of 4.5% and 46.1% respectively (P = 0.001), a shorter median progression-free survival (PFS) time of 14 ± 1.3 wk and 32 ± 2.5 wk respectively (P < 0.001), a median overall survival (OS) time of 11 ± 1.2 mo and 19 ± 1.8 mo respectively (P < 0.001), as well as in 24 patients with negative PTEN expression and in 37 patients with positive PTEN expression respectively (P < 0.001), with a responsive rate of 4.2% and 48.6% respectively, a shorter median PFS survival time of 17 ± 2.0 wk and 28 ± 1.9 wk respectively (P = 0.07), and a median OS time of 11 ± 1.3 mo and 18 ± 1.9 mo respectively (P = 0.004). Combined KRAS mutation and PTEN expression analysis showed that the PFS and OS time of patients with two favorable prognostic factors were longer than those of patients with one favorable prognostic factor or no favorable prognostic factor (P < 0.001). CONCLUSION: KRAS mutation and PTEN protein expression are significantly correlated with the response rate and survival time of Chinese mCRC patients treated with cetuximab.

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma.

BACKGROUND: The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. METHODS: Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. RESULTS: The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. CONCLUSIONS: CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.

DNA polymerase η protein expression predicts treatment response and survival of metastatic gastric adenocarcinoma patients treated with oxaliplatin-based chemotherapy.

BACKGROUND: DNA polymerase η (pol η) is capable of bypassing DNA adducts produced by cisplatin or oxaliplatin and is associated with cellular tolerance to platinum. Previous studies showed that defective pol η resulted in enhanced cisplatin or oxaliplatin sensitivity in some cell lines. The purpose of the present study was to investigate the role of pol η protein expression in metastatic gastric adenocarcinoma. METHODS: Four gastric adenocarcinoma cell lines were chosen to explore the relationship between pol η protein expression and oxaliplatin sensitivity by western blotting and MTT assay. Eighty metastatic gastric adenocarcinoma patients treated with FOLFOX or XELOX regimen as first-line chemotherapy were analyzed, corresponding pretreatment formalin-fixed paraffin-embedded tumor tissues were used to detect pol η protein expression by immunohistochemistry. Relationship between pol η protein expression and clinical features and outcome of these patients was analyzed. RESULTS: A positive linear relationship between pol η protein expression and 48 h IC50 values of oxaliplatin in four gastric cancer cell lines was observed. Positivity of pol η protein expression was strongly associated with poor treatment response, as well as shorter survival at both univariate (8 versus 14 months; P < 0.001) and multivariate (hazard ratio, 4.555; 95% confidence interval, 2.461-8.429; P < 0.001) analysis in eighty metastatic gastric adenocarcinoma patients. CONCLUSIONS: Our study indicates that pol η is a predictive factor of treatment response and survival of metastatic gastric adenocarcinoma patients treated with FOLFOX or XELOX as first-line chemotherapy. Therefore confirming the value of pol η in studies with prospective design is mandatory.

Incidence of anemia, leukocytosis, and thrombocytosis in patients with solid tumors in China.

Despite the fact that malignancies are associated with hematological abnormalities, some clinical studies have been unable to detect such a relation. The aim of our study was to detect the prevalence of pretreatment hematologic abnormalities in patients with common solid tumors and to determine if such a profile could be used for prognostic evaluations. We identified all patients in Cancer Center of Sun Yat-sen University who were diagnosed as solid tumors (breast carcinoma, hepatocellular carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, gastric cancer, cervical carcinoma, endometrial cancer, renal cell carcinoma, and non-small cell lung cancer) between January 2000 and August 2009. All subjects were investigated regarding levels of white blood cells, platelets, and hemoglobin concentration. We identified 3,180 patients with solid tumors and 285 patients with benign diseases for the final analysis. The percentages of leukocytosis, anemia, and thrombocytosis in patients with solid tumors ranged from 4.0% to 25.6%, 3.3% to 29.2%, and 2.1% to 9.7%, respectively. The multivariate Cox analysis revealed that anemia was an independent prognostic factor in patients with breast cancer (P = 0.006), hepatocellular carcinoma (P = 0.002), nasopharyngeal carcinoma (P = 0.008), and esophageal carcinoma (P = 0.001). Leukocytosis was an independent prognostic factor in patients with cervical cancer (P = 0.007). The incidence of hematological abnormalities in Chinese patients with solid tumors was relatively lower than that of the counterparts in the Western countries. A pretreatment anemia or leukocytosis can serve as a useful marker to predict outcome of patients in some of the solid tumors.

[Prognosis of 84 intrahepatic cholangiocarcinoma patients].

BACKGROUND AND OBJECTIVE: The incidence of intrahepatic cholangiocarcinoma (ICC) is low. Current treatment for ICC is unsatisfied. This study was to investigate the prognosis of patients with resectable or unresectable ICC. METHODS: Clinical data of 84 patients with pathologically confirmed ICC treated at Cancer Center, Sun Yat-sen University from January 1997 to December 2007 were reviewed. Survival and prognosis were analyzed by Kaplan-Meier method and Cox regression model. RESULTS: Of the 84 patients, 56 (66.7%) had resectable ICC, and 28 (33.3%) had unresectable ICC. Among the 56 patients with resectable ICC, 27 (48.2%) underwent radical resection, and 29 (51.8%) underwent palliative resection. The 2-year overall survival rate was 3.1% in unresectable ICC group; it was significantly higher in radical resection group than in palliative resection group (P<0.01). For the patients with resectable ICC, univariate analysis revealed that operation pattern, histological type, tumor size and number, lymph node metastasis, intrahepatic metastasis, portal vein thrombus, postoperative serum level of albumin, preoperative serum levels of CEA, CA199, TBIL, ALT and AST were related to the prognosis; multivariate analysis found that operation pattern, histological type, tumor number, preoperative serum levels of CEA, CA199 and TBIL were independent prognostic factors. For the patients with unresectable ICC, univariate analysis found that histological type and preoperative serum level of CA199 were related to the prognosis; whereas multivariate analysis found that histological type was the only independent prognostic factor. Chemotherapy showed no survival benefit in both resectable and unresectable ICC groups (P=0.30, P=0.78). CONCLUSIONS: Radical resection is the main effective treatment for ICC patients to achieve long-term survival. Preoperative serum levels of CEA, CA199 and TBIL are significant prognostic factor for patients with resectable ICC.