Dragon Mixture And Indian Strawberry Phenol Anti-tumor Effect

Cancer is a major public health problem and one of the most lethal diseases in the world, and the incidence and mortality rates are increasing in recent years.Tianlong formula is a folk recipe with traditional reputed benefits for the treatment of gynecological cancer and hepatoma.The aim of the present research is to seek the active consituents of Tianlong and investigate the underlying mechanisms of the anticancer action of the active consituents.In in vivo study,mice were implanted cervical cancer U14 cells and administrated with the aqueous extracts of Tianlong(90 g crude drug/kg),Duchesnea indica(22.5 g crude drug/kg),Solanum lytatum Thumb(22.5 g crude drug/kg) and Solanum nigrum L(22.5 g crude drug/kg).The results demonstrated that the extract of D.indica and Tianlong had similar inhibitory effect with tumor inhibition rate of 40.9%and 34.64%,respectively, other extracts did not show significantly inhibitory effect on tumor growth.And the cytotoxic activity of 15 different fractionsfrom Tianlong and its four main herbs on HeLa and SKOV-3 cells was determined using MTT assay,the fractions of Tianlong and D.indica demonstrated the most sensitive effect on cell proliferation,and the anticancer effects of Tianlong and D.indica were comparable.Based on the in vivo and in vitro findings,Duchesnea phenolic fraction(DPF) was found to be the most potent fraction in Tianlong formula,and the IC₅₀ values of DPF on HeLa and SKOV-3 cells were 57.53μg/mL and 86.12μg/mL,respectively.Cytotoxic activity of DPF on different cancer cells was determined by MTT assay.DPF at concentrations of 20-160μg/mL significantly inhibited cancer cell proliferation in a dose and time dependent manner,and showed the most sensitivity to the ovarian cancer and cervical cancer cells(IC₅₀ at 72 h were HEC-1B＞NCI-H460＞BGC-823＞SKOV-3＞CNE＞HepG2＞C33A＞HeLa).And the cytotoxic activity of DPF was characterized by tumor-selective manner,as reflected by the comparatively high IC₅₀ values on normal cells for 72 h.In mice bearing intraperitoneal U14 tumors,treatment of 0.25-1 g/kg doses of DPF significantly prolonged the survival time compared with control.In mice implanted subcutaneously cervical cancer U14 cells,administration of 0.25-1 g/kg doses of DPF markedly inhibited tumor growth in dose-dependent manner,the tumor inhibition rates of DPF were 35%,45%and 70%.Importantly,DPF showed no significant toxicity to tumor transplanted mice under this condition. AO/EB staining,DNA ladder and flow cytometry analyses revealed that DPF induced apoptosis in SKOV-3,HeLa and C33A cells evidenced by characteristic apoptotic morphological changes,nuclear DNA fragmentation and sub-G1 peak.Then the role of the molecules in apoptosis was analyzed by Western blot,RT-PCR and coloremetric assay. DPF could suppressed Bcl-2 levels,enhanced Bax levels and Bax/Bcl-2 ratio,and simultaneously translocated Bax to mitochondria followed by mitochondrial release of cyctochrome c into the cytosol and activation of effector caspase-3.Furthermore,DPF at concentrations of 20-160μg/mL arrested the cells at the S phase,with down-regulation of cyclin A,cyclin E,cyclin D1 and CDK2.Then,we use cervical cancer U14 transplanted mice to translate the in vitro findings into an in vivo animal model.The increased TUNEL-positive cells in U14 tumors of DPF-treated mice demonstrated that the anticancer effect of DPF in vivo was mediated through activation of apoptosis.Western blot and immunohistochemistry analysis showed that DPF could decrease Bcl-2 expression,increase Bax expression and Bax/Bcl-2 ratio, and accompanied by caspase-3 activation in vivo.In addition to induction of apoptosis, tumor tissues derived from DPF-treated mice showed that DPF inhibited tumor proliferation evidenced by downregulation of PCNA and ki67.Additionally,our results indicated that DPF improved function of T lymphocyte proliferation and released antibodies of B lymphocytes;decreased serum TNF-α,IL4 and IL-10;increased serum IL-2 and IFN-γ.Collectively,DPF could exert antitumor efficiency not only through reinforcing cell-mediated and humoral immune response,but also enhancing Th1-type immune response and reversing Th2 to Th1.In conclusion,we found that DPF was the active constituents of Tianlong formula.Our pronounced in vitro and in vivo studies suggested that DPF,a mixture of plant polyphenols, had potent anticancer effect and the mechanism were associated with cell cycle arrest, inhibition of cell proliferation,induction of apoptosis via mitochondrial pathway and enhancing immunological reaction.All these results provide experimental evidence to the use of Tianlong and D.indica in clinic and sustain our contention that DPF has anticancer property and merits further investigation as a potential therapeutic agent.