Anticancer Drugs. 2013 Aug;24(7):699-703.

Antiproliferative activity of the dimeric phloroglucinol and benzophenone derivatives of Hypericum spp. native to southern Brazil.

Amanda V. Pinhatti, Francisco M.C. de Barros, Caroline B. de Farias,

Gilberto Schwartsmann, Gilsane L.V. Poser, Ana L. Abujamra.

 

ABSTRACT

A large number of plants are known to possess strong antitumor properties. Previous studies have verified the antiproliferative activity of the extracts and fractions from six species of Hypericum spp. growing in southern Brazil. In the present study, the in vitro antiproliferative effects of two dimeric phloroglucinols (japonicin A and uliginosin B, isolated from Hypericum myrianthum) and two benzophenones (cariphenone A and cariphenone B, isolated from Hypericum carinatum) were investigated against three tumor cell lines (HT-29 – human colon carcinoma cells; U-251 – human glioma cells, and OVCAR-3 – human ovarian carcinoma cells). In addition, different doses of these compounds were associated with cytotoxic drugs commonly used as chemotherapy to treat these tumors (irinotecan, temozolomide and paclitaxel). Cariphenone A and cariphenone B showed moderate antiproliferative activity against all tumor cell lines at a dose of 100 μg/ml. Unlike benzophenones, japonicin A and uliginosin B exerted antiproliferative effects only in the OVCAR-3 cell line. Moreover, a very strong synergistic effect was demonstrated by the association of sub-effective doses of japonicin A with the chemotherapeutic drug paclitaxel, decreasing cellular proliferation of the OVCAR-3 cell line. These preliminary results provide a scientific basis to further pursue these compounds as potential combined therapy for certain tumor types.

PMID: 23669242

SUPPLEMENTARY

Previous studies have verified the antiproliferative activity of the extracts and fractions from six species of Hypericum spp. growing in southern Brazil [1], but we were interested in investigating which substances were responsible for this antiproliferative property. Two dimeric phloroglucinols (japonicin A and uliginosin B, isolated from the aerial parts of Hypericum myrianthum) and two benzophenones (cariphenone A and cariphenone B, isolated from the aerial parts of Hypericum carinatum) (Figures 1 and 2) were chosen for testing. The extract had already been tested on two cell lines:  HT-29 – human colon carcinoma cells and U-251 – human glioma cells. We added the OVCAR-3 cell line – human ovarian carcinoma cells to increase the breadth of the panel.

Ana Lucia Abujamra-fig1Figure 1. Flowes of the species Hypericum carinatum (1) and Hypericum myrianthum (2).

 

Ana Lucia Abujamra-fig2Figure 2. Chemical structures of the metabolites investigated: cariphenone A (1), cariphenone B (2), japonicin A (3) and uliginosin B (4).

 

The results showed that cariphenone A and cariphenone B promoted a reduction in cellular proliferation in all cancer lines at a dose of 100 mg/mL. At this concentration, higher rates of inhibition were observed against U-251 (54–53%), HT-29 (53–37%), and OVCAR-3 (32–27%) cell lines respectively, although when compared with hexane extracts from H. carinatum, these showed a moderate activity. It is possible that the compounds act synergistically in the extract, producing a major effect. This mechanism of action has already been observed, exemplified by the three benzopyrans (HP1, HP2 and HP3) isolated from H. polyanthemum [2]. Unlike benzophenones, the dimeric phloroglucinols japonicin A and uliginosin B exerted a significant effect only against the ovarian carcinoma cell line (OVCAR-3). At a dose of 50 mg/ml, both compounds reduced proliferation up to 34–35%.

Combination therapy is a common practice in cancer treatments as, besides aiming at different targets, this practice can achieve higher therapeutic effects and, consequently, reduce nonspecific side effects and resistance to drugs. The association of natural compounds with chemotherapeutic agents has been already demonstrated in other studies [3].  We investigated the association of these four natural compounds with the chemotherapeutic agents irinotecan, temozolomide, and paclitaxel which are the ones most commonly used in the clinic, among others, to treat colorectal cancer, glioma, and ovarian cancer, respectively.

Both benzophenones (1–100 mg/mL) and dimeric phloroglucinol derivatives (1–50 mg/mL) failed to show a positive interaction with irinotecan (1–10 mg/mL) and temozolomide (1–100 mg/mL). Nevertheless, an important reduction in cellular proliferation was observed in the OVCAR-3 cell line when it was treated in association with sub-effective doses of japonicin A and paclitaxel (1–10 mg/mL; Figure 3). The effects of simultaneous exposure to japonicin A and paclitaxel were analyzed using the CalcuSyn program. The sub-effective doses of paclitaxel with japonicin A in combination showed a synergistic inhibitory effect on the human ovarian cancer cell line, OVCAR-3, with a combination index (CI; concentration that produces 50% of growth inhibition) of 0.098. This suggests that paclitaxel and japonicin A, when used in combination, elicit a very strong synergistic effect. Japonicin A does not affect normal human cells obtained from primary cultures of deciduous teeth, suggesting that japonicin A may be selective for transformed cells.

We intend to study the mechanism of action by which these substances exert their antiproliferative effects, specially Japonicin A. By understanding the cellular pathways utilized by these molecules, we believe that new drugs based on benzophenones or phloroglucinol scaffolds can be a useful alternative for the treatment of this disease.

Ana Lucia Abujamra-fig3Figure 3. Associated compounds. Antiproliferative activity of subeffective doses of japonicin A associated with paclitaxel, as measured using the MTT assay. **P<0.01 when compared with controls in OVCAR-3 cell lines. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OVCAR-3, human ovarian carcinoma cells.

 

Acknowledgements

The authors are grateful to the Children’s Cancer Institute (ICI-RS) and the Federal University Hospital Research Fund (HCPA FIPE 10-0367) for financial support and by fellowships from CAPES (FMCB), CNPq (GLvP) and FAPERGS.

References

[1]Ferraz ABF, Faria DH, Benetti MN, da Rocha AB, Schwartsmann G, Henriques AT. Screening for anti-proliferative activity of six southern Brazilian species of Hypericum. Phytomedicine 2005; 12:112–115.

[2]Grivicich I, Ferraz ABF, Faria DH, Regner A, Schwartsmann G, Henriques AT, et al. Synergistic effect of three benzopyrans isolated from Hypericum polyanthemum in U-373MG glioblastoma cell line. Phytother Res 2008; 22:1577–1580.

[3] Wan XA, Sun GP, Wang H, Xu SP, Wang ZG, Liu SH. Synergistic effect of paeonol and cisplatin on oesophageal cancer cell lines. Dig Liver Dis 2008; 40:531–539.

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