| Diffuse large B-cell lymphoma(DLBCL),a subtype of non-Hodgkin’s lymphoma(NHL),is a highly heterogeneous and invasive hematological malignancy.For DLBCL,R-CHOP(Rituximab,Doxorubicin,cyclophosphamide,vincristine,and prednisone)is the main treatment option,but R-CHOP is difficult to cure in some patients.Therefore,the development of new drugs for DLBCL patients has become a research hotspot in this field.4-hydroxysalicylaniline(HDS),a natural compound,is an inhibitor of Ribonucleotide reductase(RR),which is often used in the cure of gallbladder and liver diseases.Preliminary data suggest that 4-hydroxysalicylanilide can be used to prepare drugs for the prevention and treatment of multiple myeloma and lymphoma.Its advantages include: In vitro,the compound can effectively arrest the growth of multiple myeloma and lymphoma cells;In vivo,the compound can effectively slow the growth of multiple myeloma and lymphoma in mice,and can be developed as a prophylactic and therapeutic drug for multiple myeloma and lymphoma.But the effect of the compound in DLBCL disease is still unknown.In vitro,DLBCL cell lines OCI-LY1,OCI-LY8,NU-DUL-1,U2932,and TMD8 were used to simulate DLBCL disease model.Adriamycin was commonly used as control and combination drug.To analyze the inhibitory effect of HDS(different concentration and different duration)on DLBCL tumor and its mechanism.Firstly,the effect of HDS on the proliferation of DLBCL cells was analyzed by CCK-8 method and agarose gel cloning assay.Then,the changes of DLBCL cell state during HDS administration were analyzed,such as DNA damage by γ-H2 AX staining,apoptosis by flow cytometry by Annexin V/PI(propyl iodide)staining and cell cycle arrest by flow cytometry by PI staining.Meanwhile,to analyze whether the combination of HDS and Adriamycin can arrest DLBCL tumors more effectively.The mechanism was analyzed by q PCR and Western Blot.Secondly,DLBCL cells were transplanted subcutaneously into mice to observe the proliferation of DLBCL tumors in mice.In the test group,mice were given either HDS or HDS/Adriamycin combined.In 16 days after transplantation,we recorded the survival rate,mice weight and tumor volume.16 days later,mouse were sacrificed before taking out tumor tissues,heart,liver and kidney tissues,and HE staining to analyze the structural alterations of tumor and other organs,as well as the effect of drugs on tumor and the toxic effect on mice.Finally,tumor tissue was implanted into sections for immunohistochemical staining to analyze the effects of HDS or HDS/Adriamycin on the proliferation,DNA damage and apoptosis of DLBCL cells in vivo.The results showed that HDS significantly inhibited the proliferation of DLBCL cells(OCI-LY1,OCI-LY8,NU-DUL-1,U2932 and TMD8 cell lines),and reduced the clonal formation rate of DLBCL cells.The combination of HDS and Adriamycin can arrest DLBCL tumor more effectively.HDS inhibited cell proliferation mainly by inducing G2/M phase cell cycle arrest.Moreover,it can induce significant apoptosis and DNA damage of cells,thus further inhibiting DLBCL tumor.Also,HE staining of transplanted tumor and organs of mice also showed that HDS could inhibit the proliferation of DLBCL tumor cells,induce the apoptosis of tumor cells,and thus increase the necrosis of tumor tissues,but had no dramatically effect on the tumor structure of other organs in mice,such as heart and liver.The immunofluorescence staining data of tumor tissues also showed that comparative to the control group,the expression ratio of γ-H2 AX in tumor tissues increased in HDS and HDS/ Adriamycin combined treatment group,while the ratio of Ki-67+ cells decreased,further indicating the effect of HDS on DLBCL tumor.In summary,we demonstrated that:(1)HDS can induce apoptosis and DNA damage of DLBCL cells,cause cell cycle G2/M arrest,and inhibit cell proliferation both in vivo and in vitro;(2)In combination with Adriamycin,HDS can arrest DLBCL tumor more effectively,and(3)HDS had no obvious toxicity in mice. |
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