Curcumin and its Analogues (PGV-0 and PGV-1) Enhance Sensitivity of Resistant MCF-7 Cells to Doxorubicin through Inhibition of HER2 and NF-kB Activation

Meiyanto, Edy,Putri, Dyaningtyas Dewi Pamungkas,Susidarti, Ratna Asmah,Murwanti, Retno,Sardjiman, Sardjiman,Fitriasari, Aditya,Husnaa, Ulfatul,Purnomo, Hari,Kawaichi, Masashi Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.1

Chemoresistance of breast cancer to doxorubicin is mediated mainly through activation of NF-kB and over expression of HER2. Curcumin and its analogues (PGV-0 and PGV-1) exert cytotoxic effects on T47D breast cancer cells. Suppression of NF-kB activation is suggested to contribute to this activity. The present study aimed to explore the effects of curcumin, PGV-0, and PGV-1 singly and in combination with doxorubicin on MCF-7/Dox cells featuring over-expression of HER2. In MTT assays, curcumin, PGV-0, and PGV-1 showed cytotoxicity effects against MCF-7/Dox with IC50 values of $80{\mu}M$, $21{\mu}M$, and $82{\mu}M$ respectively. These compounds increased MCF-7/Dox sensitivity to doxorubicin. Cell cycle distribution analysis exhibited that the combination of curcumin and its analogues with Dox increased sub G-1 cell populations. Curcumin and PGV-1 but not PGV-0 decreased localization of p65 into the nucleus induced by Dox, indicating that activation of NF-kB was inhibited. Molecular docking of curcumin, PGV-0, and PGV-1 demonstrated high affinity to HER2 at ATP binding site. This interaction were directly comparable with those of ATP and lapatinib. These findings suggested that curcumin, PGV-0 and PGV-1 enhance the Dox cytotoxicity to MCF-7 cells through inhibition of HER2 activity and NF-kB activation.

Natural Products for Cancer-Targeted Therapy: Citrus Flavonoids as Potent Chemopreventive Agents

Meiyanto, Edy,Hermawan, Adam,Anindyajati, Anindyajati Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.2

Targeted therapy has been a very promising strategy of drug development research. Many molecular mechanims of diseases have been known to be regulated by abundance of proteins, such as receptors and hormones. Chemoprevention for treatment and prevention of diseases are continuously developed. Pre-clinical and clinical studies in chemoprevention field yielded many valuable data in preventing the onset of disease and suppressing the progress of their growth, making chemoprevention a challenging and a very rational strategy in future researches. Natural products being rich of flavonoids are those fruits belong to the genus citrus. Ethanolic extract of Citrus reticulata and Citrus aurantiifolia peels showed anticarcinogenic, antiproliferative, co-chemotherapeutic and estrogenic effects. Several examples of citrus flavonoids that are potential as chemotherapeutic agents are tangeretin, nobiletin, hesperetin, hesperidin, naringenin, and naringin. Those flavonoids have been shown to possess inhibition activity on certain cancer cells' growth through various mechanisms. Moreover, citrus flavonoids also perform promising effect in combination with several chemotherapeutic agents against the growth of cancer cells. Some mechanisms involved in those activities are through cell cycle modulation, antiangiogenic effect, and apoptosis induction.Previous studies showed that tangeretin suppressed the growth of T47D breast cancer cells by inhibiting ERK phosphorylation. While in combination with tamoxifen, doxorubicin, and 5-FU, respectively, it was proven to be synergist on several cancer cells. Hesperidin and naringenin increased cytotoxicitity of doxorubicin on MCF-7 cells and HeLa cells. Besides, citrus flavonoids also performed estrogenic effect in vivo. One example is hesperidin having the ability to decrease the concentration of serum and hepatic lipid and reduce osteoporosis of ovariectomized rats. Those studies showed the great potential of citrus fruits as natural product to be developed as not only the source of co-chemotherapeutic agents, but also phyto-estrogens. Therefore, further study needs to be conducted to explore the potential of citrus fruits in overcoming cancer.

The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation

Meiyanto, Edy,Fitriasari, Aditya,Hermawan, Adam,Junedi, Sendy,Susidarti, Ratna Asmah 경희한의학연구센터 2011 Oriental Pharmacy and Experimental Medicine Vol.11 No.3

Tangeretin, shows cytotoxic effect on COLO 205 colon cancer cells. Combination of tangeretin with tamoxifen showed synergistic effect and increased the cancer cell sensitivity towards tamoxifen on T47D cells. However, the combination of tangeretin with chemotherapeutic agent doxorubicin on breast cancer cells have not been explored yet. Therefore, the aim of this research is to examine the improvement of cytotoxic effect of doxorubicin by tangeretin through cell death induction and cell cycle modulation on MCF-7 and T47D cells. The cytotoxic effect of tangeretin, doxorubicin, and their combination on tested cells were carried out by using MTT assay. Cell cycle distribution was determined by flowcytometer FACS-Calibur and the flowcytometry data was analyzed using ModFit LT 3.0 program. Cell death assay were done by double staining method using ethydium bromide-acridin orange. Single treatment of tangeretin 5-100 ${\mu}M$ did not show cytotoxic effect on MCF-7 and T47D cells. The combination of tangeretin 50 and 100 ${\mu}M$ with doxorubicin 200 nM (MCF-7) and 7.5 nM (T47D) increased the cytotoxic effect of doxorubicin on both breast cancer cell lines. This improvement of cytotoxic effect is due to cell death induction and cell cycle modulation. Furthermore, single treatment of tangeretin showed cell death only on T47D cell and caused G1-phase arrest on MCF-7 cell and G2/M-phase arrest on T47D cell. While doxorubicin induced cell accumulation at G2/M phase in both cancer cell lines. However, combination of tangeretin and doxorubicin increased cell death on both cancer cell lines, compared with doxorubicin by itself. The combination also showed G1-phase arrest on MCF-7 cell and increased cell accumulation at G2/M phase on T47D cell. Based on this result, tangeretin is potential to be developed as co-chemotherapeutic agent for breast cancer by inducing apoptosis and cell cycle arrest. However, the molecular mechanism need to be explored further.

The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation

Edy Meiyanto,Aditya Fitriasari,Adam Hermawan,Sendy Junedi,Ratna Asmah Susidarti 경희대학교 융합한의과학연구소 2011 Oriental Pharmacy and Experimental Medicine Vol.11 No.3

Tangeretin, shows cytotoxic effect on COLO 205colon cancer cells. Combination of tangeretin with tamoxifen showed synergistic effect and increased the cancer cell sensitivity towards tamoxifen on T47D cells. However, the combination of tangeretin with chemotherapeutic agent doxorubicin on breast cancer cells have not been explored yet. Therefore, the aim of this research is to examine the improvement of cytotoxic effect of doxorubicin by tangeretin through cell death induction and cell cycle modulation on MCF-7 and T47D cells. The cytotoxic effect of tangeretin, doxorubicin, and their combination on tested cells were carried out by using MTT assay. Cell cycle distribution was determined by flowcytometer FACSCalibur and the flowcytometry data was analyzed using ModFit LT 3.0 program. Cell death assay were done by double staining method using ethydium bromide-acridin orange. Single treatment of tangeretin 5–100 μM did not show cytotoxic effect on MCF-7 and T47D cells. The combination of tangeretin 50 and 100 μM with doxorubicin 200 nM (MCF-7) and 7.5 nM (T47D) increased the cytotoxic effect of doxorubicin on both breast cancer cell lines. This improvement of cytotoxic effect is due to cell death induction and cell cycle modulation. Furthermore,single treatment of tangeretin showed cell death only on T47D cell and caused G1-phase arrest on MCF-7 cell and G2/M-phase arrest on T47D cell. While doxorubicin induced cell accumulation at G2/M phase in both cancer cell lines. However, combination of tangeretin and doxorubicin increased cell death on both cancer cell lines,compared with doxorubicin by itself. The combination also showed G1-phase arrest on MCF-7 cell and increased cell accumulation at G2/M phase on T47D cell. Based on this result, tangeretin is potential to be developed as cochemotherapeutic agent for breast cancer by inducing apoptosis and cell cycle arrest. However, the molecular mechanism need to be explored further.

Caesalpinia sappan L. heartwood ethanolic extract exerts genotoxic inhibitory and cytotoxic effects

Edy Meiyanto,Beni Lestari,Raisatun Nisa Sugiyanto,Riris Istighfari Jenie,Rohmad Yudi Utomo,Ediati Sasmito,Retno Murwanti 경희대학교 융합한의과학연구소 2019 Oriental Pharmacy and Experimental Medicine Vol.19 No.1

Brazilin and brazilein, the major compounds of Caesalpinia sappan L. (CS) have been reported to possess antioxidant and cytotoxic activities and could potentially be used as an antigenotoxic as well as an anticancer. This study was conducted to investigate the cytotoxic and antigenotoxic effects of CS ethanolic extract (CEE). In vivo mammalian micronucleus test of CEE at the dose of 500 mg/kg BW and 1000 mg/kg BW decreased the number of MNPCE and increased the ratio of PCE to NCE meaning that CEE performed antigenotoxic effect in an in vivo model. In contrast, CEE and doxorubicin (DOXO) performed cytotoxic effect on CHO-K1 cells under MTT assay with IC50 values of 67 μg/mL and 6 μM, respectively. Interestingly, treatment of CEE in combination with DOXO and H2O2 as genotoxic inducer decreased intracellular ROS levels. In addition, in vitro genotoxicity study by using cytokinesis-block micronucleus (CBMN) assay, both of Giemsa staining and flow cytometric analysis showed that the treatment of CEE increased the number of micronuclei and correlated with apoptotic induction results. Moreover, the combination of CEE and DOXO induced cells accumulation in Sub-G1 and G2/M phase. In conclusion, CEE performed antigenotoxic effect in an in vivo model and cytotoxic effect on CHO-K1 cells.

Gynura procumbens modulates the microtubules integrity and enhances distinct mechanism on doxorubicin and 5-flurouracil-induced breast cancer cell death

Nurulita, Nunuk Aries,Meiyanto, Edy,Sugiyanto, Sugiyanto,Matsuda, Eishou,Kawaichi, Masashi 경희한의학연구센터 2012 Oriental Pharmacy and Experimental Medicine Vol.12 No.3

Recent studies both in vitro and in vivo of G. procumbens exhibits chemopreventive properties for tumor inhibition on several types of cancer. Our study was carried out to observe the anticancer property of ethyl acetate fraction of G. procumbens leaves (FEG) on breast cancer cells as well as the co-chemotherapeutic potential, and to investigate its molecular mechanisms. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the growth inhibitory effect of FEG, doxorubicin (DOX), and 5-fluorouracil (5-FU) and their combination. Flowcytometry, 4',6-diamidino-2-phenylindole (DAPI) staining, and immunobloting were used to explore the mechanism of cell cycle arrest and apoptosis. FEG inhibited cell proliferation, induced G1 phase arrest and apoptosis. The inhibitory effect of FEG was enhanced when combined with Dox and 5-FU. The apoptosis induction was related to the increase of c-PARP expression after combination treatment of FEG and Dox or 5-FU onMCF-7 cells. However, treatment of DOX, 5-FU, and FEG on T47D cells, resulting no significance DNA fragmentation and nuclei condensation evidance. Only combination treatment of 5-FU+FEG showed c-PARP expression in T47D cells. In T47D cells, The FEG treatment also caused the decrease of microtubule expression as shown by Western blotting assay. The decreasing level of microtubul expression might be caused by protein aggregation, as shown by immunostaning using ${\alpha}$-tubulin antibody. All these results suggest that FEG potentiates the DOX and 5-FU efficacy on MCF-7 and T47D cells. FEG induces T47D cell death through different mechanism than MCF-7 that proposed to be mitotic catastrophe. The FEG may have specific targeted on microtubule integrity modulation leading to the cell cycle arrest and proliferation inhibition. Further FEG could be developed as a co-chemotherapeutic agent for reducing side effect and have specific molecular target for breast cancer.

Combination of Potassium Pentagamavunon-0 and Doxorubicin Induces Apoptosis and Cell Cycle Arrest and Inhibits Metastasis in Breast Cancer Cells

Putri, Herwandhani,Jenie, Riris Istighfari,Handayani, Sri,Kastian, Ria Fajarwati,Meiyanto, Edy Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.5

A salt compound of a curcumin analogue, potassium pentagamavunon-0 (K PGV-0) has been synthesized to improve solubility of pentagamavunon-0 which has been proven to have anti-proliferative effects on several cancer cells. The purpose of this study was to investigate cytotoxic activity and metastasis inhibition by K PGV-0 alone and in combination with achemotherapeutic agent, doxorubicin (dox), in breast cancer cells. Based on MTT assay analysis, K PGV-0 showed cytotoxic activity in T47D and 4T1 cell lines with $IC_{50}$ values of $94.9{\mu}M$ and $49.0{\pm}0.2{\mu}M$, respectively. In general, K PGV-0+dox demonstrated synergistic effects and decreased cell viability up to 84.7% in T47D cells and 62.6% in 4T1 cells. Cell cycle modulation and apoptosis induction were examined by flow cytometry. K PGV-0 and K PGV-0+dox caused cell accumulation in G2/M phase and apoptosis induction. Regarding cancer metastasis, while K PGV-0 alone did not show any inhibition of 4T1 cell migration, K PGV-0+dox exerted inhibition. K PGV-0 and its combination with dox inhibited the activity of MMP-9 which has a pivotal role in extracellular matrix degradation. These results show that a combination of K PGV-0 and doxorubicin inhibits cancer cell growth through cell cycling, apoptosis induction, and inhibition of cell migration and MMP-9 activity. Therefore, K PGV-0 may have potential for development as a co-chemotherapeutic agent.

Immunomodulatory Effects of Hexane Insoluble Fraction of Ficus septica Burm. F. in Doxorubicin-treated Rats

Nugroho, Agung Endro,Hermawan, Adam,Nastiti, Kunti,Suven, Suven,Elisa, Pritha,Hadibarata, Tony,Meiyanto, Edy Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.11

The use of chemotherapeutics induces cardiotoxicity and affects immune functions, therefore development of combinatorial agents against cardiotoxicity and immunosuppression needs to be explored. Previous studies of the hexane insoluble fraction (HIF) of an ethanolic extract of Ficus septica leaves showed anticancer effects singly and in combination with doxorubicin on T47D breast cancer cells. In this present study, it was evaluated for its immunomodulatory activities in doxorubicin-treated rats. Thirty male Sprague Dawley rats were divided into five groups consisting of six rats each as follows: Group 1, receiving oral saline 10 ml/kg BW (control group); Group 2, receiving HIF dose 750 mg/kg BW orally, once daily; Group 3, receiving HIF dose 1.500 mg/kg BW orally, once daily; Group 4, given oral saline 10 ml/kg BW (normal group); Group 5, receiving HIF dose 1.500 mg/kg BW orally, once daily. The rats of group 1-3 were intramuscularly administered with doxorubicin at a dose of 4.67 mg/kg BW at the days 1 and 4 to suppress immune functions. Concomitantly, the rats were treated with saline or HIF for seven consecutive days (1 to 7). Treatment of HIF succeeded in reducing side effects of doxorubicin based on increasing lymphocyte density and phagocytosis activity and capacity of macrophages, as well as increasing the CD8+ blood level and decreasing spleen IL-10 expression. Hexane insoluble fraction of of ethanolic extract of Ficus septica leaves has potential as a protective agent combined with doxorubicin.