The main objective of this study was to examine the activity of artemisinin as an anti-cancer treatment of the urinary bladder cancer. Where, the biochemical results revealed that urea and creatinine activities were significantly increased in groups treated with BBN, BBN + cisplatin and BBN + artemisinin + cisplatin compared to the control group. While, urea and creatinine levels were non-significant in groups treated with artemisinin alone and BBN + artemisinin. In similar previous studies, [21] reported that the treated normal mice and breast cancer-bearing mice with artemisinin did not show any significant alterations in both creatinine and urea levels. In the findings of [22], using cisplatin, as chemotherapeutic, has induced nephrotoxicity which leads to increasing of both creatinine and blood urea nitrogen levels. In another study, [23] has found that artesunate (derivative of artemisinin) treatment, caused strongly down-regulated the creatinine and blood urea nitrogen levels in cisplatin induced acute kidney injury in mice. Our findings also revealed that using artemisinin has improved the side effects caused by BBN and cisplatin, and reduced the urea and creatinine levels in bladder cancer bearing mice.
The hematological results showed that HB and RBCs counts were significant decrease in both BBN cancer group and BBN + cisplatin treated group compared to the control, while PLT and WBCs were significant decrease only in BBN + cisplatin treated group. In contrast, PLT and WBC showed significant increase in BBN cancer group. However, these findings revealed that HB, PLT, RBC and WBC counts were improved in BBN (bladder cancer group) and BBN + cisplatin treated group, as a result of artemisinin treatment. In a previous study, [21] found that there were no-significant alterations in HB, RBCs and WBCs counts in normal mice treated with or without artemisinin. While, the breast tumor-bearing mice showed reduction in HB and RBCs counts, concomitant with an increase in the number of WBCs count. However, the author concluded that all these aberrations were reverted to normal values upon the treatment by artemisinin. In the findings of [24], in the breast cancer, high dose of artesunate (derivative of artemisinin) and gemcitabine obviously affected on the hematological markers WBC, RBC, PLT and HGB compared to the control group. The authors revealed that these treatments did not cause obvious injury and inflammation in the artesunate treated mice. [25] reported that single dose of cisplatin injection caused significant decrease in the mean values of Hb, PLT, RBCs and WBCs compared to the control groups. The authors have observed significant reduction in first two weeks of cisplatin injection, and on subsequent intervals the values showed a meagre gradual improvement. This study has proved that the beneficial effects of gallic acid in ameliorating of hematological alterations induced by cisplatin in Wistar albino rats, in addition, possible preventive role of gallic acid against cisplatin induced toxicity. Our findings agree with the previous studies, where we found that HB, PLT, RBCs and WBCs counts are improved after treatment with artemisinin alone and in combination with cisplatin in mice urinary bladder cancer.
The results of the histopathological examinations of kidney tissues showed interstitial nephritis with the presence of lymphoplasmacytic interstitial infiltrates, the tubules revealed cloudy swelling and glomerular mesangial hypercellularity in the BBN group. While in BBN group treated with cisplatin, glomeruli with mesangial hypercellularity, glomerular sclerosis and moderate to severe interstitial inflammation were observed. On the other hand, mild interstitial inflammation and no vascular changes were observed in the BBN group treated with artemisinin. While, cloudy swelling of tubules with casts and interstitial inflammation were observed as a result of treatment with artemisinin plus cisplatin in mice bladder cancer. In a previous study, [26] found that histopathological examination of the kidneys and testes showed no evidence of structural abnormalities in rats treated with artemisinin only on doxorubicin-induced renal and testicular toxicity. Results of [23] showed that artesunate (derivative of artemisinin) improved renal dysfunction in cisplatin-induced acute kidney injury, and the results demonstrated that the artesunate significantly decreased renal damage. However, our findings support the previous studies, where artemisinin ameliorated glomerular mesangial hypercellularity and lymphoplasmacytic interstitial infiltrates in BBN-induced urinary bladder cancer. Moreover, artemisinin combined with cisplatin significantly decreased renal damage caused by cisplatin in urinary bladder cancer bearing mice.
Concerning to the histopathological examinations of bladder tissues, an invasive urothelial carcinoma with an invasive nest within the lamina propria was seen in BBN group. While, urothelial dysplasia and a focus of CIS were seen in BBN group treated with cisplatin. On the other side, moderate urothelial dysplasia and a non-invasive papillary urothelial neoplasm were observed in BBN group treated with artemisinin. While, focal dysplastic changes in the urothelium were found in BBN group treated with both artemisinin and cisplatin. In a previous study, [15] has explained that treatment with artesunate (derivative of artemisinin) at doses of 20, 100, and 200 mg/kg significantly reduced tumor sizes in a dose-dependent manner. Rats in groups 100 mg/kg and 200 mg/kg showed a lower incidence of high-grade tumors than those in BBN group. This finding suggests that the artesunate may have potential as a bladder cancer treatment. In another study, [27] has found that artesunate significantly inhibited tumor cell growth and proliferation in a time- and dose-dependent manner in cisplatin-sensitive, and cisplatin-resistant bladder cancer cells. In another similar study, [28] has declared that the artesunate exhibited significant anti-tumor effect on lung cancer A549 cells and this efficiency could be enhanced by combination with cisplatin. The combination of artesunate and cisplatin showed promising anti-tumor activity against head and neck cancer cell lines, even at low cisplatin doses, according to [29]. However, the previous studies agree with and confirm our findings. Where, the artemisinin exhibited significant anti-cancer effect on mice urinary bladder cancer and this efficiency could be increased by combination with cisplatin.
Concerning FGFR3, HRAS, P53 and KDM6A genes expression results, FGFR3 and HRAS (oncogenes) showed strongly down-regulated expression levels in both BBN and BBN + cisplatin groups treated with artemisinin, while, almost half down-regulated expression levels were shown in BBN group treated with cisplatin alone. In contrast, P53 and KDM6A (tumor suppressor genes) showed strongly up-regulated expression levels in BBN and BBN + cisplatin groups which treated with artemisinin. While, slightly up-regulated expression levels were observed in BBN group treated with cisplatin alone. In a previous study, [30] has demonstrated that dihydroartemisinin (derivative of artemisinin) caused an apoptosis in non-small cell lung cancer cells with EGFR or RAS mutation in-vitro and in-vivo. [31] found that after cisplatin treatment intermediate and low levels of FGFR3 and FGFR4 were expressed in all five head and neck squamous cell carcinoma cell lines. Where, tumor growth or neoangiogenesis, as angiogenesis serves is a critical role in tumor growth, and the inhibition of this process via cisplatin alone is insufficient. In another study, [32] reported that pterostilbene further decreased tumor growth in mice that received cisplatin and gemcitabine of human bladder cancer with oncogenic HRAS. Where, pterostilbene is safe and readily available food ingredient in edible plants worldwide. In the findings of [6], the salirasib (salicylic acid derivative that inhibits Ras protein activation) and siRNA induced HRAS knockdown produced tumor suppressive effects regardless of HRAS mutational status in bladder cancer cell lines. Proteomic analysis revealed that several metabolic pathways were significantly down-regulated in bladder cancer cells treated with salirasib. [33] studied the effect of the combination of artesunate and WNT974 (selective and orally bioavailable inhibitor). The author found that significantly exhibited synergistic effect in reducing colorectal cancer growth via down-regulation of KRAS (belongs to the RAS oncogenic family) levels.
In a previous study to [34], KDM6A and its function are often lost in bladder cancer as a result of inactivating mutations. KDM6A-deficient cells are dependent on the function of another protein, called Enhancer of Zeste Homolog 2 (EZH2), which is often over-expressed and associated with poor prognosis in muscle-invasive bladder cancer. Therefore, EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in multiple mouse models. In another related study, [35] has also reported that a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF (Switch/Sucrose Non-Fermentable) mutations to EZH2 inhibition alone and in combination with cisplatin treatment. However and according to the findings in our study, we suggest that EZH2 inhibition could be enhanced via artemisinin alone and in combination with cisplatin treatment.
For p53, in a previous study to [36], Artemisia annua L. extract (source of artemisinin) enhanced up-regulating of p53 and p63 expression levels with promising in vivo anticancer effect. Thus, authors suggest that Artemisia annua is used as a complementary therapy in breast cancer. In another study, [37] has indicated that artemisinin up-regulated p53 expression through the ERK1/2 signaling pathway. The author also revealed that artemisinin exhibits direct anti-proliferative actions on gastric cancer cells, and it might be a good candidate for clinical trial in gastric cancer treatment. Results of [38], dihydroartemisinin induces death receptor 5 (DR5) expressions through regulating p53 with the most pronounced enhancement of DR5 expression in colon carcinoma cell lines. Eventually, [28] reported that artesunate induced effectively apoptosis and worked cooperatively with cisplatin in lung cancer cell lines. However, an evident exchange in p53, Bax and Bcl-2 in the dual-drug-treated cells was observed, which is correlated with the intensive incidence of apoptosis.