He-Chan Pian inhibits the metastasis of non-small cell lung cancer via the miR-205-5p-mediated regulation of the GREM1/Rap1 signaling pathway.

BACKGROUND: He-Chan Pian (HCP), a traditional Chinese medicinal formula, shows promising efficacy for the treatment of lung cancer. PURPOSE: Gremlin (GREM1) plays an important role in gastrointestinal tumor metastasis; however, little is known about its role in lung cancer. We determined the mechanism underlying the protective effect of HCP against metastasis in a mouse model of non-small cell lung cancer (NSCLC) and demonstrated the role of GREM1. METHODS: Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the herbal components and metabolites from the serum of HCP-treated mice. The tumor, liver, and kidney were examined histologically, and the antitumor effects and toxicity of HCP were evaluated. Levels of epithelial-mesenchymal transition (EMT)-associated transcription factors were measured using western blotting in tumors from five groups (i.e., model, HCP [L], HCP [M], HCP [H], and positive control [cisplatin, DDP]). Differentially expressed proteins and genes were identified using protein chip and sequencing analyzes, respectively. Short hairpin RNAs and overexpression plasmids were introduced into cells to evaluate the effects of GREM1. To evaluate proliferation, migration, and invasion, the expression levels of proteins involved in the Rap1 pathway and EMT were measured in vitro. Xenograft tumors with overexpression-GREM1 (OE-GREM1) in A549 cells were examined for cell proliferation. A dual-luciferase assay was performed to verify the direct interaction of GREM1 with miR-205-5p in lung cancer. RESULTS: Thirty-six ingredients and bioactive constituents detected in the serum of HCP-treated mice were identified as the key compounds involved in the inhibition of tumor growth. Animal experiments revealed that HCP significantly decreased tumor volumes and had no adverse effects on the liver or kidney or side effects. GREM1 upregulation was closely related to tumor metastasis and was regulated by miR-205-5p, as confirmed using a dual-luciferase reporter assay. OE-GREM1 promoted A549 cell migration and invasion, promoted EMT, and increased the expression of Rap1 pathway intermediaries, whereas shGREM1 had the opposite effects. Furthermore, the effects of OE-GREM1 on proliferation in the A549 xenograft mouse model were attenuated, although HCP has an inhibitory effect on tumors. CONCLUSION: Our results suggest that HCP contributes to the inhibition of NSCLC metastasis via the Gremlin/Rap1 signaling pathway regulated by miR-205-5p.

Arenobufagin Promoted Oxidative Stress-Associated Mitochondrial Pathway Apoptosis in A549 Non-Small-Cell Lung Cancer Cell Line.

Arenobufagin (ARE) has demonstrated potent anticancer activity in various types of tumor, but the role and mechanism of ARE for lung cancer remain unclear. Oxidative stress exists under normal conditions and is an inevitable state in the body. A variety of noxious stimuli can break the equilibrium state of oxidative stress and promote apoptosis. Here, we used a CCK-8 assay to examine cell viability. We determined oxidative stress damage by measuring levels of intracellular ROS and levels of GSH, SOD, and MDA. Annexin V-FITC/PI double staining assay, as well as the Hoechst 33258 staining, was used to detect ARE-induced apoptosis in A549 cell. Evaluation of the expression level of the specified molecule was indicated by Western blot and qRT-PCR. Loss of function experiment was carried out using NAC pretreatment. The experimental results show that ARE significantly declines in the viability of A549 cells and increases the apoptosis rate of A549 cells. As reflected in cell morphology, the A549 cells showed features of shrinkage and had incompletely packed membranes; the same phenomenon is manifested in Hoechst 33258 staining. Following ARE treatment, the ROS level in A549 cells was rising in a concentration-dependent manner, and so were MDA and GSH levels, while the SOD level was decreasing. Moreover, we found that ARE can decrease mitochondrial membrane potential (MMP), and a cascade of apoptotic processes can be triggered by decreased MMP. Importantly, we found significant changes in protein expression levels and mRNA levels of apoptosis-related proteins. Furthermore, when we used NAC to restrain oxidative stress, the expression levels of apoptosis-related proteins have also changed accordingly. Our data demonstrate that apoptosis in the non-small-cell lung cancer (NSCLC) cell line A549 is caused by oxidative stress due to ARE. Our research also shows that ARE may have the potential to become a targeted therapeutic for the treatment of NSCLC in the future.

In Vitro and In Vivo Inhibitory Effect of Gujin Xiaoliu Tang in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a serious threat to people's health. This study aims to determine the possible effect of Gujin Xiaoliu Tang (GJXLT) on NSCLC, which is an empirical formula from Professor Dai-Han Zhou. In this study, chromatographic fingerprinting of GJXLT and A549 cell model in vitro and in vivo was established. We cultured A549 cells in vitro and found that GJXLT inhibited A549 cell growth and induced apoptosis. Compared with the control group, the expression of p-STAT3 and VEGF proteins in the GJXLT groups was decreased. Similar findings were also observed in vivo. First, GJXLT inhibited the growth of transplanted tumor and did not reduce the weight of the tumor-bearing mice in comparison with that of the control group. Then, the Ki-67 expression of transplanted tumor in the GJXLT groups was decreased. In addition, the apoptosis rate of transplanted tumor in the GJXLT groups was increased. Overall, our data showed that GJXLT inhibited A549 cell proliferation and induced apoptosis in vivo and in vitro. Furthermore, GJXLT inhibited the growth of lung cancer xenograft in nude mice model with no obvious side effects. The anti-tumor effect of GJXLT might also be related to the inhibition of p-STATS and VEGF expression in the JAK2/STAT3 pathway. Our results demonstrated the potential of GJXLT as a novel treatment for NSCLC.

Expression of prolyl 4-hydroxylase beta-polypeptide in non-small cell lung cancer treated with Chinese medicines.

OBJECTIVES: To investigate the role of prolyl 4-hydroxylase beta polypeptide (P4HB) expressed in lung carcinoma and the intervention effect of Yiqi Chutan Formula (, YQCTF). METHODS: Lung carcinoma model was established by subcutaneously inoculating LEWIS lung carcinoma cells in C57BL/6J mice. The differential expression of P4HB protein between the YQCTF (3.0 g/kg, gavage, once daily, 21 days) group and the control group was acquired by a 2 fluorescence difference gel electrophoresis (2D-DIGE), verified by Western blotting and identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). The expression of P4HB and P4HB mRNA in cultured A549 cells from cisplatin (DDP) 1.5 µg/mL group and 15% serum combined with DDP 1.5 µg/mL group were detected by cellular immunohistochemistry and reverse transcription-polymerase chain reaction, respectively. RESULTS: The proteomics research discovered that one-third of differential proteins including P4HB were decreased in the YQCTF group (P<0.01). Clinical pathology and tissue microarray studies showed that P4HB expression in lung cancer tissue was stronger than adjacent tissues and normal lung epithelial (P<0.01). In the YQCTF and DDP combined groups, the expression of P4HB and P4HB mRNA in A549 cell were decreased significantly (P<0.01). CONCLUSION: YQCTF could inhibit the LEWIS lung carcinoma's growth, decrease the expression of P4HB in LEWIS lung carcinoma and A549 cells. YQCTF might take effect through regulating P4HB in endoplasmic reticulum to inhibit the incidence and growth process of lung carcinoma.

Effectiveness of acupuncture for palliative care in cancer patients: a systematic review.

OBJECTIVE: To critically evaluate the currently available randomized clinical trials regarding the effectiveness of acupuncture in palliative care for cancer patients, hence, to provide sufficient evidences for the widespread use of acupuncture in cancer treatment. METHODS: Two independent reviewers extracted data from all of the randomized clinical trials (RCTs) that assessed the efficacy of acupuncture in palliative care for cancer patients. Seven databases were searched from their respective inception to December 2010. All eligible trials identified were evaluated by two independent reviewers using the Jadad scale, and data from the articles were validated and extracted. RESULTS: In total, 33 RCTs met the inclusion criteria. The effects of acupuncture on different cancer-related aspects were shown, including chemotherapy or radiotherapy-induced side effects (13/33, 39.4%), cancer pain (6/33, 18.2%), post-operative urinary retention (4/33, 12.1%), quality of life (2/33, 6.1%), vasomotor syndrome (2/33, 6.1%), post-operative gastrointestinal dysfunction (2/33, 6.1%), prevention of prolonged postoperative ileus (2/33, 6.1%), joint symptoms (1/33, 3.0%), and immunomodulation (1/33, 3.0%). CONCLUSIONS: The result of our systematic review suggested that the effectiveness of acupuncture in palliative care for cancer patients is promising, especially in reducing chemotherapy or radiotherapyinduced side effects and cancer pain. Acupuncture may be an appropriate adjunctive treatment for palliative care.

Effects of yiqi chutan tang on the proteome in Lewis lung cancer in mice.

In order to verify effects of yiqi chutan tang on lung cancer and assess molecular mechanisms involved we focused on size, tumor weight and the numbers of lung metastases and differential expression protein spot information acquired by two-way fluorescence with a tumor difference gel electrophoresis (2D-DIGE) system, and differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF). Differences were finally verified by Western blot and fluorescence quantitative PCR. We found that tumor size, tumor weight in yiqi chutan tang treatment group were significantly less than that in model group (p<0.01), with a tumor growth inhibition rate of 57.2%. For gel diagram analysis of 2D-DIGE system, compared with model group, there were 44 expressed differentially protein spots, of which 6 were up-regulated and 38 were down-regulated. Among these proteins, 37 (30 down-regulated and 7 up-regulated) were successfully identified by MALDI-TOF-TOF. In conclusion, yiqi chutan tang effects on Lewis lung cancer appeared highly related to down-regulated expression of Hspd1, prolyl 4-hydroxylase, protein disulfide-isomerase A3 precursor, EG433182, heat shock protein 5 precursor, heat shock protein 9 and stress-induced phosphoprotein 1.

[Apoptosis inducing effect of Hechanpian on human lung adenocarcinoma A549 cells].

OBJECTIVE: To study the apoptosis inducing effects of Hechanpian (HCP) on human lung adenocarcinoma A549 cells. METHODS: HCP containing rat serum was prepared and applied on A549 cells. The cell growth inhibition rate was tested by MTT assay; the effect of HCP on cell apoptosis was observed with Propidium iodide (PI) staining and flow cytometry analysis; the mRNA expression of epidermal growth factor receptor (EGFR) was detected through RT-PCR. RESULTS: The growth of A549 cells was obviously inhibited after being treated by HCP containing serum, and the cells presented an apoptotic change. The cell apoptosis rate after treated by serum containing 10% and 20% HCP was 20.5% and 33.2%, respectively, significantly higher than that in the control (6.1% in cells didn't treated with HCP, P < 0.05). Compared with control, EGFR mRNA expression in HCP treated cells was significantly lower (P < 0.05). CONCLUSION: HCP has apoptosis inducing effect on A549 cell, and its molecular mechanism is probably correlated with the inhibition of EGFR gene transcription.

[Proteomics in Chinese medicine researches: current status, problems and strategies].

Proteomics plays important roles in Chinese medicine research at post-genomics era. Its research idea and methods are beneficial for elucidating some elemental features of Chinese medicine. At present, Chinese medicine proteomic studies are mainly focusing on the syndromatology and medical herbal pharmacology. However, there are still some problems, the most important matter was that most of the results were merely the superficial delineations. Further research should put emphasize on the unremitting and penetrating study of proteomics, molecular biology and bioinformatics integrally for illuminating Chinese medicine theory deeply to promote the modernization of Chinese medicine research.

[Effect of traditional Chinese medicine in improving quality of life of patients with non-small cell lung cancer in late stage].

OBJECTIVE: To observe the effect of traditional Chinese medicine (TCM) in improving quality of life of patients with non-small cell lung cancer (NSCLC) in III or IV stage, for establishing TCM therapeutic regimen on late NSCLC. METHODS: A total of 294 patients in 6 hospitals were randomly assigned into three groups, 99 in the TCM group treated with TCM according to disease and syndrome differentiation, 92 treated with chemotherapy in the western group and 103 treated with combined therapy of TCM and chemotherapy in the integrative group. Six items, including physical status, social/family status, intercourse with physicians, emotional status, functional status and additional concerning status, were investigated and analyzed by using Functional Assessment of Cancer Therapy-lung (FACT-L). RESULTS: The scores of social/family status and intercourse with physicians were insignificantly different in all three groups before and after treatment (P > 0.05). The improvement of physical status in the TCM group, and that of emotional status, functional status and additional concerned status in the integrative medicine group were superior to those in the other groups (P< 0.05). CONCLUSION: TCM has certain antagonistic effect on the adverse reaction of chemotherapy, and it can improve the quality of life of patients to certain extent.

[Prognostic analysis of stage III-IV non-small cell lung cancer patients treated by traditional chinese medicine].

BACKGROUND & OBJECTIVE: Chemotherapy is a treatment for stage III-IV non-small cell lung cancer (NSCLC), but the efficacy is not ideal. Traditional Chinese medicine (TCM) has certain effect on NSCLC. This study was to investigate various factors that affect the prognosis of advanced NSCLC, and evaluate the role of TCM in enlonging survival time of patients with stage III-IV NSCLC. METHODS: The NSCLC patients who meet the inclusive criteria were randomized into TCM group, combination (TCM plus NP regimen) group, and chemotherapy group, and received relevant treatments. The median survival time (MST) was calculated by Kaplan-Meier method. The prognosis of the patients was analyzed by COX regression method. RESULTS: A total of 294 stage III-IV NSCLC patients were enrolled, of which 99 were in TCM group, 103 in combination group, 92 in chemotherapy group. The MST were 292 days in TCM group, 355 days in combination group, and 236 days in chemotherapy group; the cumulative survival rates were 45.38%, 48.86%, and 42.17%, respectively (P>0.05). Cox regression analysis indicated that therapy, gender, disease course, erythrocyte sedimentation, KPS score, tumor size, and patient's weight were independent prognostic factors of stage III-IV NSCLC. CONCLUSION: Compare with chemotherapy alone, TCM combined with chemotherapy may prolong the survival time of stage III-IV NSCLC patients.

[Analysis on the prognostic factors in patients with large hepatocarcinoma treated by shentao ruangan pill and hydroxycamptothecine].

OBJECTIVE: To observe the effect of intervention therapy with Shentao Ruangan pill (SRP) and hydroxycamptothecine (HCPT) in treating 85 patients with middle-advanced large hepatocarcinoma, and to analyze the factors that could affect the prognosis. METHODS: Eighty-five patients were randomly divided into the treated group (n = 52) and the control group (n = 33). The treated group was treated by oral taking of SRP combined with local perfusion of HCPT through hepatic artery catheterization, while to the control group, the conventional therapy, transcatheter arterial chemoembolization (TACE) was conducted for control. The clinical efficacy of treatment in the two groups was evaluated by the change of tumor size, the factors related with prognosis were analyzed using Cox proportional hazards model and the analysis of survival conducted by Kaplan-Meier method. RESULTS: (1) The tumor size reducing rate in the treated group was 19.2% and the tumor size stabilizing rate was 82.7%, while those in the control group was 21.2% and 81.8% respectively, comparison of the criteria between the two groups showed insignificant difference (P > 0.05); (2) The median survival time, 0.5- year, 1- year and 2- year survival rate in the treated group was 326 days, 80.95%, 41.39% and 12.42% respectively, those in the control group was 262 days, 64.29%, 25.00% and 8.33% respectively, comparison between the two groups showed significant difference (P < 0.05); (3) Among the 3 TCM types in patients, the survival time and rates in patients of Gan-excess with Pi-deficiency type was similar to those in patients of Gan-heat with blood stasis type showing insignificant difference (P > 0.05), but as compared with those in patients of Gan-Shen Yin-deficiency type, the difference was significant (P < 0.05) ; (4) Beneficial factor to the prognosis were therapeutic method, that used in the treated group was superior to that used in the control group. The risk factors to the prognosis were TCM type, clinical stage and liver function. Patients of Gan-excess with Pi-deficiency type had the optimal prognosis, those of Gan-heat with blood stasis type the next and of Gan-Shen Yin-deficiency the worst. The later the clinical stage and the worse the Child-Pugh grade of liver function was, the worse the prognosis would be. CONCLUSION: (1) SRP combined with HCPT intervention treatment is superior to the simple TACE treatment in elevating patients' survival rate and time; (2) There are some relations between TCM types and prognosis; (3) Local Chinese drug therapy combined with systemic therapy could be one of the effective measures of non-operational therapy in treating large hepatocarcinoma.

[Analysis of short-term therapeutic efficacy of integrated traditional and Western medicine in treating non-small cell lung cancer].

OBJECTIVE: To observe the short-term therapeutic efficacy of integrated traditional and Western medicine (ITWM) in treating non-small cell lung cancer (NSCLC) in III and IV phase. METHODS: Adopting the prospective, multi-centered, randomized and controlled method for clinical research, 324 patients who conformed to the enrolling standard were divided by ratio of 1:1:1 into the Chinese medicine (CM) group (n= 99), the ITWM group (n 03) and the Western medicine (WM) (n=92) group. The excluded or dropping off cases were 10 in CM, 6 in ITWM and 14 in WM. Clinical trials were conducted in 6 hospitals and 3 months of treatment was taken as one therapeutic course. The main observation indexes were tumor size, Karnofsky scores, body weight, adverse reaction, etc. RESULTS: The total effective rate of tumor remission in the 3 groups was 4.0%, 26.2%, and 14.1%, respectively, statistical significance was shown in the difference among them (chi = 21.72, P = 0.000 < 0.017). The total tumor stabilization rate was 66.7 , 81.6%, and 76.1 , respectively, by rectification test, no significance was shown in difference among them (chi2 = 6.052, P = 0.049 > 0.017). Karnofsky scoring showed that after 90 days of treatment, Karnofsky score raised in the CM and ITWM group, but lowered in the WM group, paired t-test showed significant difference in the ITWM group before and after treatment. The Karnofsky score in IWTM was higher than that in CM and WM with significant difference (H = 10.572, P = 0.000 < 0.05). The patients' body weight in the 3 groups were all reduced. The reduction in the CM and ITWM group was lower than that in WM group, among which, significant difference was shown in CM and WM group when compared with the same group before treatment (P < 0.05). The effect in the ITWM and CM group was better than that in WM group in aspects of improving such tumor related symptoms as cough, short breath, anorexia, fatigue, etc. Observation of adverse reaction showed that lesser hemotoxicity of III and IV grade appeared in the CM and ITWM group than that in the WM group, and significant difference was shown in counts of white blood cells, granulocytes, platelets hemoglobin, etc. among the 3 groups (P < 0.01). CONCLUSION: ITWM therapy showed better short-term efficacy in treating patients with NSCLC than CM or WM alone, showing the superiority of ITWM therapy. It can be adopted as an effective therapeutic program with low-toxicity.