Dihydroartemisinin induces apoptosis of cervical cancer cells via upregulation of RKIP and downregulation of bcl-2

Abstract

Treatment of recurrent and metastatic cervical cancer remains a challenge, especially in developing countries, which lack efficient screening programs. In recent years, artemisinin and its derivatives, such as dihydroartemisinin (DHA), which were traditionally used as anti-malarial agent, have been shown to inhibit tumor growth with low toxicity to normal cells. In this study, we investigated mechanisms underlying the anti-tumor effect of DHA in cervical cancer. We evaluated the role of DHA on the expression of bcl-2 and Raf kinase inhibitor protein (RKIP), which is a suppressor of metastasis. The MTT assay was used to compare the proliferation of untreated and DHA-treated Hela and Caski cervical cancer cells. Flow cytometry was used to determine the percentage of cells at each stage of the cell cycle in untreated and DHA-treated cells. We used RT-PCR and western blots to determine the expression of bcl-2 and RKIP mRNA and proteins. We evaluated the effect of DHA treatment in nude mice bearing Hela or Caski tumors. DHA-treated cells showed a time- and dose-dependent inhibition of proliferation and a significant increase in apoptosis. The expression of RKIP was significantly upregulated and the expression of bcl-2 was significantly downregulated in DHA-treated cells compared with control cells. DHA treatment caused (1) a significant inhibition of tumor growth and (2) a significant increase in the apoptotic index in nude mice bearing Hela or Caski tumors. Our data suggest that DHA inhibits cervical cancer growth via upregulation of RKIP and downregulation of bcl-2.

Keywords: Raf kinase inhibitor protein; apoptosis; bcl-2; cervival cancer; dihydroartemisinin; metastasis; tumor.

Figure 1. DHA inhibits proliferation of Hela and Caski cells in a dose- and time-dependent manner. (A) Hela cells were treated with different doses of DHA for 48 h. (B) Caski cells were treated with different doses of DHA for 48 h. (C) Hela cells were treated with 20 μmol/L of DHA for varying amounts of time (D) Caski cells were treated with 20 μmol/L of DHA for varying amounts of time.

Figure 2. DHA-treated Hela and Caski cell line were subjected to AO/EB fluorescent staining. (A) Morphology of Hela and Caski cell treated with 20 μM treatment of DHA for 48 h after AO/EB fluorescent staining. (1) Hela Control; (2) Hela DHA; (3) Caski Control; (4) Caski DHA. Up arrow, NV; star, VA; circle, NVA; triangle, NVN. (B) Bar representation of AO/EB fluorescent staining. *P < 0.05 indicates a significant difference between control and DHA groups for each cell line.

Figure 3. DHA effects on cell cycle profiles of untreated and DHA-treated Hela and Caski cells. *P < 0.05 indicates a significant difference between control and DHA groups for each cell line. ^†P < 0.05 indicates a significant difference between Hela and Caski control groups. ^‡P < 0.05 indicates a significant difference between Hela and Caski DHA groups.

Figure 4. mRNA expression of RKIP and Bcl-2 in untreated and DHA-treated Hela and Caski cells. (A) Representative electrophoresis images; (B) mRNA expression of RKIP normalized to that of GAPDH; (C) mRNA expression of bcl-2 normalized to that of GAPDH. *P < 0.05 indicates a significant difference between control and DHA groups for each cell line.

Figure 5. Protein expression of RKIP and Bcl-2 in untreated and DHA-treated Hela and Caski cells. (A) Representative images of western blots protein gels; (B) Protein expression of RKIP normalized to that of GAPDH; (C) Protein expression of bcl-2 normalized to that of GAPDH. *P < 0.05 indicates a significant difference between control and DHA groups for each cell line.

Figure 6. HE staining of tumors in vivo. (A) HE staining of tissue from untreated Hela tumors (100×); (B) HE staining of tissue from untreated Hela tumors (400×); (C) HE staining of tissue from DHA-treated Hela tumors (100×); (D) HE staining of tissue from DHA-treated Hela tumors (400×); (E) HE staining of tissue from untreated Caski tumors (100×); (F) HE staining of tissue from untreated Caski tumors (400×); (G) HE staining of tissue from DHA-treated tumors (100×); (H) HE staining of tissue from DHA-treated Caski tumors (400×). Arrows show focal hemorrhage and necrosis at the center of the tumor in control groups.

Figure 7. HE staining of important organs and Wright–Giemsa staining of bone marrow in nude mice. (A) HE staining of kidney; (B) HE staining of liver; (C) HE staining of heart; (D) HE staining of bone marrow (100×).

Figure 8. Effect of DHA on cell death in Hela and Caski cell lines. (A) TUNEL-staining of untreated and DHA-treated Hela and Caski cells. (B) Apoptotic index of untreated and DHA-treated Hela and Caski cells. *P < 0.05 indicates a significant difference between control and DHA groups for each cell line.