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Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (5): 425-438.doi: 10.1016/S2095-4964(14)60045-5

• Research Article • Previous Articles     Next Articles

Low doses of ethanolic extract of Boldo (Peumus boldus) can ameliorate toxicity generated by cisplatin in normal liver cells of mice in vivo and in WRL-68 cells in vitro, but not in cancer cells in vivo or in vitro

Jesmin Mondala, Kausik Bishayeea, Ashis Kumar Panigrahib, Anisur Rahman Khuda-Bukhsha   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India
    Fisheries and Aquaculture Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India
  • Received:2014-03-23 Accepted:2014-07-08 Online:2014-09-10 Published:2014-09-15

Objective

Use of cisplatin, a conventional anticancer drug, is restricted because it generates strong hepatotoxicity by accumulating in liver. Therefore its anticancer potential can only be fully exploited if its own toxicity is considerably reduced. Towards this goal, ethanolic extract of the plant, Boldo (Peumus boldus), known for its antihepatotoxic effects, was used simultaneously with cisplatin, to test its ability to reduce cisplatin’s cytotoxicity without affecting its anticancer potential. 

Methods

The cytotoxicity of Boldo extract (BE) and cisplatin, administered alone and in combination, was determined in three cancer cell lines (A549, HeLa, and HepG2) and in normal liver cells (WRL-68). Drug-DNA interaction, DNA damage, cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP, ΔΨ) were also studied. Hepatotoxicity and antioxidant activity levels were determined by alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and glutathione assays in mice. The cytotoxicity of related proteins was tested by Western blotting. 

Results

Co-administration of BE and cisplatin increased viability of normal cells, but had no effect on the viability of cancer cells. Boldo protected liver from damage and normalized different antioxidant enzyme levels in vivo and also reduced ROS and re-polarized MMP in vitro. Bax and cytochrome c translocation was reduced with caspase 3 down-regulation. Further, a drug-DNA interaction study revealed that BE reduced cisplatin’s DNA-binding capacity, resulting in a reduction in DNA damage. 

Conclusion

Results indicated that a low dose of BE could be used beneficially in combination with cisplatin to reduce its toxicity without hampering cisplatin’s anticancer effect. These findings signify a potential future use of BE in cancer therapy.

Key words: Drug-related toxicity, Peumus boldus, Plant ethanolic extract, Cytotoxicity, Liver, Anti-oxidant

Figure 1

Cell viability assay by MTTCell viability was checked by MTT with different doses of cisplatin and BE on different cell lines. A and B: Cisplatin (A) and BE (B) treatment on WRL-68 and HepG2 cell lines. C: Co-treatment of cisplatin and BE on WRL-68, HepG2, HeLa and A549 cells. Data are represented as percentage of control and are presented as mean±standard error of mean. **P<0.01 (for WRL-68) versus untreated control and △△P<0.01 (for HepG2) versus untreated group was considered statistically significant. MTT: thiazolyl blue tetrazolium bromide."

Figure 2

Drug-DNA interaction studyCD spectral analysis of DNA-binding ability of cisplatin and BE alone and co-administration. CD: circular dichroism; BE: Boldo extract."

Figure 3

Morphological analysis (A) The microscopic study of different cancer cells under binocular phase contrast microscope (Leica, Germany, ×40) revealed that cisplatin could induce cellular structural distortion. Co-treatment of BE and cisplatin did not show any cellular structural reformation. Cisplatin also induced damage to the normal liver cells (WRL-68). BE co-treatment with cisplatin could prevent the damage caused by cisplatin (CisP (cisplatin), 20 μmol/L; BE1, 48 μg/mL; and BE2, 64 μg/mL for in vitro). (B) 4′,6-Diamidino-2-phenylindole staining indicates that there was damage in DNA when cisplatin was administered on both HepG2 and WRL-68 cells. Co-administration of BE reduced the damage induced by cisplatin in WRL-68 cells but not in HepG2 cells."

Figure 4

DNA damage and apoptosis analysis DNA-gel assay of normal cells (A, WRL-68) and cancer cells (C, HepG2) reveals that cisplatin treatment in both normal and cancer cells induced DNA damage. Co-administration of Boldo extract (BE) reduced damage in DNA of normal cells but not in cancer cells. Annexin V/PI assay indicates apoptosis induction when cisplatin was administered on both WRL-68 cells (B) and HepG2 cells (D). Co-administration with BE reduced apoptosis induced by cisplatin in normal cells but there was no visible effect in cancer cells. In B and D, X-axis denotes annexin V-fluorescein isothiocyanate and Y-axis denotes propidium iodide (PI). LN1=untreated, LN2=64 μg/mL BE, LN3=20 μmol/L cisplatin, LN4=20 μmol/L cisplatin+64 μg/mL BE. The quadrants of lower left, lower right, upper right and upper left show the percentage of live (annexin-ve; PI-ve), early apoptotic (annexin+ve; PI-ve), late apoptotic (annexin+ve; PI+ve) and necrotic cells (PI +ve) respectively."

Figure 5

Cell cycle analysis (A) Cell cycle analysis indicates that increase in number of sub-G cells with reduction in S-phase population, while cisplatin was administered. But BE treatment along with cisplatin reduced DNA damage and increased DNA synthesis in normal WRL-68 cells [M1=Sub-G, M2=G0/G1, M3=S, M4=G2/M]. Y axis denotes counts of cells. (B) GSH depletion in WRL-68 cells (LN1=control, LN2=BE2, LN3=cisplatin, LN4=cisplatin+BE1, LN5=cisplatin+BE2). Data are represented as percentage of control and are presented as mean ± standard error of mean. Statistical significance was considered as **P<0.01, vs untreated control. (C) Protein expression study by Western blot: cytosolic and mitochondrial Bax and cytochrome c activity were analyzed, along with caspase 3 activity. GAPDH and VDAC1 served as loading control for cytosolic and mitochondrial fraction, respectively. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; VDAC1: voltage-dependent anion-selective channel protein 1."

Figure 6

Microscopical and flow-cytometrical examining for ROS estimation in both HepG2 and WRL-68 cells In normal cell line, cisplatin treatment increased ROS generation compared to the control, but co-administration of BE with cisplatin reduced this ROS generation, whereas in cancer cell lines treatment with cisplatin alone as well as cisplatin plus BE increased ROS generation compared to control. ROS: reactive oxygen species; BE: Boldo extract."

Figure 7

Microscopical and flow-cytometrical checking for MMP depolarization in both HepG2 and WRL-68 cells MMP: mitochondrial membrane potential, BE: Boldo extract."

Figure 8

Effects on the survival time, GSH and pathologic change (A) Survivability curve of cancerous mice receiving drug treatment as compared to the control. Carcinogen-treated mice receiving no treatment survived less than those which received drug treatment. This plot shows the survivability of mice in last three months after cancer induction. (B) Cisplatin could introduce damage to the DNA of the cancerous liver tissue. BE co-treatment did not show any ameliorative effect on histological features of cancerous liver tissue. In the normal mice, when cisplatin was injected, it induced damage to the liver cells, but when BE was co-treated with cisplatin, the damage was minimum (CisP (cisplatin), 10 mg/kg bw; BE1, 20 mg/kg bw; and BE2, 40 mg/kg bw). (C) GSH depletion in mice liver. LN1=control, LN2=BE2 (40 mg/kg bw), LN3=cisplatin (10 mg/kg bw), LN4=cisplatin+BE1 [cisplatin (10 mg/kg bw) + BE1 (20 mg/kg bw)], LN5=cisplatin+BE2 [cisplatin (10 mg/kg bw)+BE2 (40 mg/kg bw)]. Data are represented as percentage of control and are presented as mean±standard error of mean. Statistical significance was considered as **P<0.01 versus untreated control."

"

Figure 9

Schematic representation of action of Boldo extract in reducing cytotoxicity generated by cisplatin in liver cells in vitro and in vivo ALT: alanine aminotrasferase; AST: aspartate aminotrasferase; GSH: redused glutathione; LDH: lactate dehydrogenase; MMP: mitochondrial membrance protential; ROS: reactive oxygen species."

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