| BackgroundHead and neck squamous cell carcinoma(HNSCC) develops from the mucosal linings of the upper aerodigestive tract, comprising oral cavity, nasal cavity, paranasal sinu ses, larynx, and pharynx, and is the sixth most common malignancy. Despite that combined therapy currently applied in the treatment of HNSCC, have partially improved clinical outcomes, the overall 5-year overall survival rate of advanced stage(T3 and T4) remains embarrassing. Therefore, new therapeutic targets and modalities for effective chemoprevention and treatment are required to improve the survival of patients with HNSCC.Signal transducers and activators of transcription(STATs) proteins are a famil y plays an essential role in initiation of malignant transformation and cancer progression. STAT3, a signal transducer and an oncogene as well, is identified as a protein that closely associated with oncogenesis of malignant tumors. STAT3 is a latent cytoplasmic protein that can be activated by various extracellular polypeptides, including cytokines, growth factors, and oncogenes, etc. These stimuli interact with specific cell surface receptors, resulting in phosphorylation of specific Tyrosine or Serine on STAT3 protein, which in turn induces STAT3 monomer’s homodimerization and/or heterodimerization through reciprocal SH2-phosphotyrosine interaction and translocate to the nucleus. There they bind specific DNA and direct expression of downstream genes and proteins associated with cell survival and proliferation, cell cycle, apoptosis, and angiogenesis.Under physiological conditions, the activated STAT3 is inactivated within a short time due to dephosphorylation and relocates to cytoplasm. By contrast, STAT3 is persistently activated in over a dozen types of human cancers, including all the major carcinomas as well as some hematologic tumors. A wide array of studies reported that in tumour cells, persistent phosphorylation of STAT3 can promote acquisition of the ability to proliferate uncontrollably, resist apoptosis, sustain angiogenesis and evade immune surveillance, which leads to an intractable even lethal outcome of cancer patients. Conversely, therapeutic blockage specifically targeting STAT3 activation can convert the efficacy of STAT3 phosphorylation from pro-cancer to anti-cancer events. To this end, several putative agents have been identified as inhibitor of STAT3 phosphorylation and studied both in vitro and in vivo, but none of them was used in clinical trials and practical use in cancer treatment for ambiguous after-effects to host and other properties.The sesquiterpene lactone dihydroartemisinin(DHA), a semisynthetic derivative of a Chinese medicinal herb—Artemisia annua and main active metabolite of the artemisinin, is one of most effective antimalarial drugs used for treating malignant malaria and cerebral malaria. In recent years, an extensive body of evidence from studies has suggested that DHA possess antitumor activity and selective cytotoxicity to various malignancies. The anti-cancer effects of DHA have been proposed to be mediated through apoptosis induction, cell cycle arrest, and anti-angiogenesis. However, little is known about the effects of DHA on upstream mediators and signaling pathways of apoptosis, cycle arrest, and c in HNSCC.Objective1. To evaluate the effects of DHA on the cell proliferation, cell cycle, apoptosis, and migration of three types of HNSCC cells.2. To test the possibility of DHA as a putative STAT3 inhibitor and validate the underlying mechanisms of the inhibition.3. To investigate the synergistic efficacies of DHA and chemotherapy drug cisplatin on HNSCC cells.4. To estimate the effects of DHA on the tumor growth in the xenograft animal models of human HNSCC.MethodsThe study includes three parts.Part one: The effects of DHA on the HNSCC cells proliferation, cell apoptosis, cell cycle, migration and angiogenesis. Fadu, Cal-27 and Hep-2 cells were treated with various concentrations of DHA for 24 or 48 h, and cell viability was tested via MTT assay. Then, IC50 values of DHA were calculated for the three cell lines, and cell apoptosis and cell cycle were also measured by flow cytometry upon treatment with DHA. Moreover, migration was observed by wound healing assay. In further study, effects of DHA on expression of a few of proteins, all of which are important regulators of cell proliferation, apoptosis, cell cycle, migration and angiogenesis, were determined by Western blot.Part two: The study of underlying mechanisms of antiproliferative effects of DHA to HNSCC cells. After exposing Fadu, Cal-27 and Hep-2 cells to different concentrations of DHA or DMSO, the expression of proteins p-STAT3, p-Jak2, p-Src, p-EGFR, p-ERK1/2 and p-Akt were analyzed by Western blotting. Subsequently, the three HNSCC cells were treated with DHA for 24 h and exposed to IL-6(20ng) for 1 h, and levels of p-Jak2 and p-STAT3 were determined by Western blotting. In further study, Cal-27 cells were transfected with dominant negative EGFR(DN-EGFR), DN-Jak2, DN-Src, constitutive active STAT3(CA-STAT3), or empty vector, and exposed to 80 μM DHA for 24 h. Expression of p-EGFR, p-Jak2, p-Src and p-STAT3 were studied by Western blotting. Moreover, the three tumor cell lines were exposed to different concentrations of DHA, AZD1480 and AG490 or DMSO for 24 h, after which time the expression levels of p-Jak2 and p-STAT3 were analyzed by Western blotting. Last, Fadu, Cal-27 and Hep-2 cells were treated with either DHA or cisplatin, or a combination of both for 24 h, and proliferation inhibition was determined by MTT assay.Part three: The effects of DHA on the xenograft animal tumor models. Cal-27 cells were used to establish xenograft tumors in BALb/c mice, and animals were treated with DHA(50mg/kg) or DMSO. In entire animal experiments, behavior of animals, body weight of nude mice and mean tumor volume were observed. After 28 days of exposure to DHA, the animals were sacrificed, and the tumors were excised and weighed. In further study, the upstream and downstream proteins of STAT3 signaling pathway in xenograft tumors were measured by Western blotting. Expression of p-Jak2 and p-STAT3 in tumor tissues were also evaluated by immunohistochimistry. These data from in vitro and in vivo experiments provide the rationale for the further clinical evaluation of DHA as a chemotherapeutics for treatment of HNSCC patients.ResultsPart one: DHA inhibited proliferation, growth and migration of HNSCC cells in vitro.1. DHA inhibited the proliferation of all three HNSCC cells. Compared with other two kinds of tumor cells, Cal-27 and Hep-2 cells, Fadu cells were the least sensitive to the compound. Dose-dependent apoptosis induction and G0/G1 cell cycle arrest were attributed to the inhibitory effect of DHA on the three HNSCC cells. Further, DHA also reduced the migration of all three cell lines in a time-dependent manner.2. After various concentrations of DHA treatment, inhibition of Mcl-1, Bcl-x L, Cyclin D1, VEGF, MMP-2 and MMP-9 occurred in the three HNSCC cell lines.Part two:Inhibition of STAT3 activation by DHA in HNSCC cells was attributed to selective blockade of Jak2 phosphorylation.1. Under normoxia or hypoxia, DHA inhibited p-STAT3(Tyr705) activation in the three HNSCC cell lines in dose- and time-dependent manners, but did not alter the status of p-STAT3(Ser727) phosphorylation.2. After treating Fadu, Cal-27 and Hep-2 cells with different concentrations of DHA, inhibition of Jak2 activation was prominent, but little or no inhibition of Src, EGFR, Akt and ERK phosphorylation was observed. Moreover, DHA abolished the p-Jak2 and p-STAT3 induced by IL-6 in HNSCC cells.3. Cal-27 cells were successfully transfected with DN-EGFR, DN-Jak2, DN-Src, or CA-STAT3 constructs. The four transfected-cell lines exposed to DHA treatment for 24 h, and western blotting analysis was used to assess the expression of p-Jak2, p-EGFR, p-Src and p-STAT3. The data showed that treatment with DHA decreased the expression of p-Jak2 and p-STAT3, but had little effects on p-EGFR and p-Src expression. Surprisingly, DHA did not result in reduction of STAT3 activation in CA-STAT3-expressing Cal-27 cells, and CA-STAT3 construct attenuated the effect of apoptosis and G0/G1 cell cycle arrest induced by DHA. In contrast, treatment with DHA resulted in inactivation of p-STAT3 and increase of G0/G1 cell cycle arrest and apoptosis in empty vector cells.4. Levels of p-Jak2 and p-STAT3 were decreased by DHA, AZD1480 and AG490 in HNSCC cells in a dose-dependent manner.5. Significant decrease in proliferation was observed in Cal-27 and Hep-2 cells treated in a combination of DHA(10 μM) with cisplatin(10 μM), as well as DHA(20 μM) with cisplatin(5 or 10 μM). In Fadu cells, synergistic effects were noted when cells were cultured with 10 or 20 μM DHA and 5 or 10μM cisplatin. The data showed that DHA synergistically potentiates the antitumor activity of cisplatin in HNSCC cellsPart three:DHA inhibited tumor growth in the xenograft of human HNSCC cells.Compared to the vehicle-treated control group, administration of DHA(50mg/kg) for 28 days significantly decreased tumor size, overall tumor weight and mean tumor volume. Nevertheless, the body weight of nude mice under DHA treatment did not changed dramatically. Furthermore, consistent with the results in vitro, upstream and downstream proteins of STAT3 signaling pathway including p-STAT3, p-Jak2, Mcl-1, Bcl-x L, Cyclin D1 and VEGF were suppressed in DHA-treated tumors, but p-ERK1/2, p-Akt, p-Src and p-EGFR did not changed remarkably.Conclusion1. The antiproliferative effects of DHA, including inhibition of cell viability and migration, as well as increase of G0/G1 cell cycle arrest and apoptosis, were associated with downregulation of p-STAT3 and its encoded and regulated proteins Mcl-1, Bcl-x L, Cyclin D1, VEGF, MMP-2 and MMP-9 in HNSCC cells.2. Selective inhibition of STAT3 phosphorylation at Tyr705 by DHA in HNSCC cells is attributed to specific blockade of Jak2 phosphorylation. Notably, DHA did not altered the level of overexpressed p-STAT3, apoptosis, and cell cycle in Cal-27 cells transfected with CA-STAT3 construct, suggesting that DHA interfere with the functions of STAT3 at its pre-activation stage, and inactivation of Jak2/STAT3 cascade play a critical functional role in DHA-mediated G0/G1 cell cycle arrest and apoptosis in HNSCC cells.3. Concordantly with the effects of AZD1480 and AG490, two specific inhibitors of Jak2/STAT3 signal pathway, DHA remarkably downregulated levels of p-Jak2 and p-STAT3 in HNSCC cells as well. However, DHA did not alter the level of overexpressed Akt and ERK phosphorylation. The data implicated that DHA is a novel STAT3 inhibitor that could effectively block Jak2/STAT3 signaling pathway.4. DHA synergistically potentiated the antitumor activity of cisplat in in HNSCC cells possibly through inhibition of STAT3 activation, resulting in reversion of the cell cycle distribution patterns, including accumulation of G0/G1 phase and decrease of S and/or G2 phase.5. DHA regimen significantly decreased xenograft tumor size, overall tumor weight, and resulted in downregulation of upstream and downstream proteins of STAT3, including p-Jak2, Mcl-1, Bcl-x L, Cyclin D1, VEGF, MMP-2 and MMP-9. The observation further confirmed the notion that inactivation of Jak2/STAT3 si gnaling pathway play a pivotal role in growth-inhibitory effects of DHA toward HNSCC cells. |
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