| Objective:To assess the efficacy of patients diagnosed as DLBCL with Benefiting Vital Energy and Eliminating Phlegm Prescription Combined with chemotherapy as first-line treatment. sTLC was detected in this group of patients, to determine its predicting and prognostic value in untreated DLBCL and to observe its change induced by Benefiting Vital Energy and Eliminating Phlegm Prescription.To observe anti-tumor activity and mechanism of Benefiting Vital Energy and Eliminating Phlegm Prescription on DLBCL in vitro and in vivo, and to determine the role of Benefiting Vital Energy and Eliminating Phlegm Prescription in the regulation of AKT/mTOR signaling pathway in this preliminary study.Methods:A total of 46 patients with DLBCL were enrolled in the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (GZUCM) and the cancer centre of SunYat-senUniversity between March 2014 and May 2015. Patients in the The First Affiliated Hospital of GZUCM were treated with the combination of Benefiting Vital Energy and Eliminating Phlegm Prescription and CHOP±R regimen(the experimental group), while cases in the cancer centre of SunYat-senUniversity were treated with CHOP+R regimen (the control group). Serum samples were collected at the time of inclusion before any chemotherapy and after the 1st to the 4th cycles of chemotherapy that made a total of 5 times of blood specimen collection. sTLC (κ and λ), IgG, IgA, and IgM concentrations were tested by nephelometry. The differences of the treatment effects and the changes of serum indexes were compared between the experimental and control groups, and the relationship between the serum indexes and prognosis were analyzed.The human DLBCL lymphoma cell lines OCI-ly10 and SUDHL-6 were cultured and passaged. CCK-8 method was used to observe the inhibitory effect of Benefiting Vital Energy and Eliminating Phlegm Prescription on tumor cell growth. Flow cytometry (annexin V-FITC/PI) staining was used to detect the role of Benefiting Vital Energy and Eliminating Phlegm Prescription in inducing apoptosis in DLBCL cell. The expression of AKT pAKT, pS6, pPRAS40 and pGSK on DLBCL in cell line was assessed by Western blot method. We also created a tumor bearing BALB/c-nu nude mice model of DLBCL, to observe the tumor volume, tumor weight and to calculate the tumor inhibition rate. In addition, protein expression belonging to the Akt/mTOR signaling pathway was detected by immunohistochemical method.Result:The most frequent syndrome in DLBCL was intermin-gled phlegm and blood stasis, accounting for 64% of all patients. The total objective response rate (ORR) was 80.4%, with a 2-year OS and PFS were 92.7% and 79.6%, respectively. There was no significant difference between the experimental group and control group in terms of ORR, OS and PFS. Compared with the control group, leukopenia in the experimental group were more modest (P=0.048). STLC test found that the serum kappa light chain increased in 19.6% of patients, and the serum level of lambda light chain increased in 17.4% of patients, respectively. Significant correlation between kappa light chain levels and multiple organ involvement was found (P=0.01). Both lambda light chain (P=0.029) and kappa and lambda light chain (P=0.002) are adverse prognostic factors and are related to the decrease of OS. Further analysis of influence of the sTLC on PFS found that kappa light chainlevels increase is associated with a decline in PFS (P=0.044). Multivariate analysis showed that elevated kappa light chain levels are independent prognostic indicators of PFS (P=0.031). In the analysis of the change trend of sTLC, kappa and lambda light chain, kappa chain and lambda chain curve decreased during the chemotherapy (P<0.001), reaching the lowest point after the 4th cycle of chemotherapy. Compared to the control group, the experimental group had a more remarkable effect on decreased lambda light chain curve (P=0.041).Cell experiments showed that Benefiting Vital Energy and Eliminating Phlegm Prescription can inhibit the growth of DLBCL cell line SUDHL-4, SUDHL-6 and OCI-ly10, and this kind of inhibition had a certain time and concentration dependency. In flow cytometry, the apoptosis rate of low level, median level and high level Benefiting Vital Energy and Eliminating Phlegm Prescription group were increased compared with the negative control group, and stronger effect to promote tumor cell apoptosis was found in the high level group than the negative control group. Western blot results showed that Benefiting Vital Energy and Eliminating Phlegm Prescription can significantly reduce expression of p-AKT, p-S6, pPRAS40 and pGSK in DLBCL cell lines. Animal Experimental results show that tumor volume and tumor weight of the TCM group, the adriamycin group and the combination group were lower than those in the control group (P<0.05). Furthermore, the tumor inhibition rate of combination group (47.3%) was higher than that of TCM group (26.4%) and adriamycin group (31.2%). Differences of pAKT, pGSK, pS6 and pPRAS40 protein expression among the TCM and the control groups were significant (P=0.05;P=0.011;P=0.004; P=0.02, respectively).Gonelusion:In this study, we found that DLBCL patients with Benefiting Vital Energy and Eliminating Phlegm Prescription Combined with with CHOP+R chemotherapy as first-line treatment can reduce the toxicity of bone marrow suppression. STLC detection and clinical characteristics analysis found that serum kappa light chain is related to the decrease of OS in DLBCL patients and is affecting PFS as an independent prognostic indicators. The changes of serum kappa and lambda light chain during chemotherapy indicated that sTLC could reflect the changing trend of tumor load. Compared with the control group, the effect of the decrease of the kappa and the light chain curve was more significant, which indicated the possibility of more decrease of the tumor load in the Benefiting Vital Energy and Eliminating Phlegm Prescription combined with CHOP+R chemotherapy group.Cell and animal experiments found that Benefiting Vital Energy and Eliminating Phlegm Prescription combined with with CHOP+R chemotherapy can inhibit the growth of DLBCL cell lines, which has a certain time dependence and concentration dependence, and its mechanism is related to the promotion of tumor cell apoptosis. Benefiting Vital Energy and Eliminating Phlegm Prescription may play an important role in promoting apoptosis through reducing the expression of key proteins including pAKT, pS6, pPRAS40 and pGSK in the AKT/mTOR signaling pathway. |
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