INTRODUCTION:
Cancer is characterized by uncontrolled growth of a given cell type together with the invasion of surrounding tissue and spread to malignant cells1. Breast cancers (mostly) begin either in the breast tissue made up of glands for milk production, called lobules, or in the ducts that connect the lobules to the nipple. The remainder of the breast is made up of fatty, connective, and lymphatic tissues2.
American Cancer Society reported in 2017, an estimated 252,710 new cases of invasive breast cancer will be diagnosed among women and 2,470 cases will be diagnosed in men. Approximately 40,610 women and 460 men are expected to die from breast cancer in 2017. About one-third of postmenopausal breast cancers are thought to be caused by behavioral factors that are modifiable, such as postmenopausal obesity, physical inactivity, use of combined estrogen and progestin menopausal hormones, alcohol consumption, and not breastfeeding, endocrine factors, including early menarche, nulliparity, and also Hormone Replacement Therapy application, followed by genetic and life style factors. A previous study demonstrated that the 7,12-dimethyl benz (a) anthracene (DMBA) was able to induce squamous cell carcinomas in the hamster buccal pouches3. DMBA is commonly used as potent organ and site specific carcinogen, causes metabolic activation produces the ultimate carcinogen, dihydrodiol epoxide by over production of ROS4.
Primary cancer treatments have incorporated both chemotherapeutic agents and ionizing radiation to eliminate the bulk of the tumour mass. However, these treatments cause cancer relapse and even destroy healthy cells. The use of conventional cancer treatment also causes the development of drug resistance in tumour cells. Therefore, potential anticancer drugs need to be identified for anticancer from alternative sources, including natural products5. Tuber rodent (Typhonium flagelliforme (Lood) Bl) claimed to cure some cancers including breast cancer, testicular cancer, prostate cancer, colon cancer, bone cancer, lung cancer and liver cancer, and also have the ability as anti proliferation or positive activity in apoptosis. T. flagelliforme ethanolic extract has shown anticancer potential through the expression of telomerase in Raji cells (reduced the expression of telomerase)6. A previous study demonstrated that the fraction of T. flagelliforme inhibited leukemia cancer-cell proliferation selectively, via the induction of apoptosis7. It was known that some of plant fractions inhibit the growth of lung cancer cells NCL-H23 and fraction of dichlorome than gave IC50 7.5 µg/mL and also known to induce apoptosis8. In addition, the chemical compounds in T. flagelliforme also inhibits the growth of breast cancer cells HS578T and the component of this plant known as pheophorbides which has an activity of antiproliferation increases by photoactivity9. Other studies mentioned the ethanolic extract of T. flagelliforme have cytotoxic potential against MCF-7 cells with IC50 5.59µg/mL10.
However, less information is available about in vivo study of cancer chemopreventive activity from this plant. Therefore, the present study was aimed to investigate chemopreventive activity from rodent tuber (T. flagelliforme). The survival rate of animals, latency period of tumors, percentage of tumor incidence, number and size of tumors, weight of tumors, fluorescence intensity of autofluorosphore and histological evaluation were also determined.
MATERIALS AND METHODS:
Animals:
Female Sprague-Dawley rats with naive and healthy condition were used in this present study (43 days old, weight of 80–100g). Prior to treatment, animals were acclimatized for 7 days and given food and water, weight was measured and behavior was observed. Animals that did not show a behavioral abnormalities and variations in weight of <10% during the acclimatization were prepared to be treated.
Plant and extract preparation:
Typhonium flagelliforme (Lood) Bl was identified in School of Science and Biological Technology, Bandung Institute of Technology, Bandung, Indonesia. T. flagelliforme (CV. Herbagold Purwakarta-Indonesia) was extracted using maseration with 95% ethanol and condensed using rotary evaporator.
Experimental design:
Rats were given of Typhonium flagelliforme extract orally for 7 days before and after induction of DMBA single dose (15mg/rats). DMBA was purchased from Sigma and was dissolved on 1mL sesame oil11. Rats given only DMBA were used as a control group. The observation take about 20 weeks after the induction. The investigation of cancer chemopreventive effect was performed using T. flagelliforme extract with dose of 50 mg/kg bw (as described previously)10. Parameters measured were survival rate of animals, latency period of tumors, percentage of tumor incidence, number of tumors and tumor weight. All the obtained data was analyzed using independent t student (p < 0.05).
Measurement of fluorescence intensity of autofluorosphore (FIA):
The blood was collected from tail vein using heparinized eppendorf tube and then was centrifuged at 3000rpm for 5 min to separate plasma, afterward the pellets were washed with 0.9% saline and were centrifuged at 3000 rpm (procedure was repeated thrice to seperate erythrocytes). A known amount of erythrocytes were lysed with cold distilled water for 60 min at 0⁰C. After that, the erythrocytes were centrifuged at 15,000rpm for 20 min to separate the erythrocyte membrane. Next, 2 mL of analytical grade acetone was added then was vortexed and centrifuged at 5000rpm for 10 min. The clear supernatants (acetone extracts) were subjected to fluorescent spectral analyses at 400 nm excitation using a spectrofluorometer (Shimadzu RF-540) and the emission spectra was scanned from 430nm to 700nm12. The data obtained was analyzed using independent t student (p < 0.05).
Histological evaluation:
At the ending observation, a small amount of tumor tissue of rats were fixed in 10% phosphate-buffer formalin solution and were embedded in paraffin, then were stained with hematoxylin-eosin. Observations were carried out with a light microscope with 100 x magnification. The level of malignancy was determined based on grading system by Scarff-Bloom-Richardson (SBR)13,14.
RESULTS AND DISCUSSION:
The survival rate of animals until ending observation exhibited that whole animal of chemopreventive group were survived. The latency period of tumors appearance was 12 weeks, which is longer or 3 weeks delayed compared to the control group (9 weeks) after induction. The percentage of tumor incidence until ending observation in the test group was 33.33, this result indicated that the T. flagelliforme extract reduced incidence of tumor that appears on each rat, significantly, compared to the control group (100%) (refer to Figure 1).
Figure 1: Percentage of tumors incidence of 50 mg/kg bw T. flagelliforme extract group (TF 50) and control group. *Significantly different compared to control (p < 0.05)
The others parameters (number and size of tumors, weight of tumors, FIA values) were exhibited differences result from the test group (given T. flagelliforme extract) compared to the control group, respectively (refer to Figure 2).
Figure 2: The average of all parameters measurement per rat of 50 mg/kg bw of T. flagelliforme extract group (TF 50) compared to control group.
*Significantly different compared to control (p < 0.025)
The number of tumors per rat that appears in the test group were range between 0–2 tumors with an average of 0.50, this result indicated significantly different compared the number of tumors in the control group of 1–4 tumors per rat with an average of 2.83 (p < 0.025). The tumors weight of the test group were average 0.26g compared to the control group was 5.34g per rat. The size of tumors were measured at ending observation, after rats were sacrificed, the tumors were taken away and measured. The size of tumors were exhibited that significant decrease in the test group (0.39cm2) compared to the control group (2.4cm2).
The fluorescence intensity of autofluorosphore (FIA) was carried out at 20 weeks after induction by taking erythrocytes samples of each rat from each groups. The FIA is one of parameters that used to seeing fruitfulness of cancer therapy. A previous study presented that the FIA on tumor or cancer condition will increase as the malignancy12. As shown in Figure 2, the measurements of FIA values generated from test group was average 6.00 compared to control group was average 11.62. These results indicated that the group given T. flagelliforme extract was significant different compared to the control group, statistically.
The results demonstrated that the group given T. flagelliforme extract was capable to suppress the amount and growth of tumors in animals test compared to the control group, significantly. It has been known that T. flagelliforme capable to withstand the growth of several types of cancer cells including HS578T breast cancer cells through in vitro studies8. The compounds in the fraction of this plant is also thought to be apoptosis and antiproliferation against breast cancer cells9. In addition, previous study demonstrated that rodent tuber extract (RTE) capable to inhibit cell proliferation of human breast cancer primary T47D cells and provide information that the use of RTE alone is better than the combination with tamoxifen (TAM) against T47D cells15.
Apoptosis is a process of cell death programmed and is a natural mechanism in the human body to kill cells that are considered damaged to maintain growth and health of multicellular organisms. However, in the case of cancer cells, the mechanism of this nature can be avoided because cancer cells have mutated so that can not be recognized by the body. Therefore, this mechanism in cancer treatment can be used as approaches to inhibit the development or kill the cancer cells. According to the previous report, T. flagelliforme plants have the ability to induce apoptosis of human T4 lymphoblastoid (CEMss) cell line to interfere with its DNA cells cleavage. In addition, it was reported that T. flagelliforme possess a valuable antileukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis7. The apoptosis process of T. flagelliforme was expected by changing the morphology of cancer cells by interfering with DNA division of cancer cells so that occurrence the condensation of chromatin which then causes cells blebbing. The histopathology figure was also shown the allegedly mitotic cells that ready to regenerated and growth (refer to Figure 3).
Figure
3: Histopathology figures of T. flagelliforme extract (a) and control
group (b). Mitotic cells shown by arrow sign ( 
)
Mitotic cells can be used as a parameter of cancer malignancy, because the more numbers of mitotic cells the greater chance of cancer cells to grow and develop, and the higher level of malignancy. Scarf-Bloom-Richardson has categorized the level of cancer based on tubular formation, nuclear pleomorphism, and mitotic count, and based on this classification, the malignancy level of control group was categorized in Grade 2 (Moderate-differentiated breast cancer) or moderate malignancy. Meanwhile, as shown in Figure 3, the test group (given T. flagelliforme extract) was categorized in Grade 1 tumors (well-differentiated breast cancer) or low malignancy. According to the previous report, chemopreventive activity may be antioxidant/free radical-scavenging constituents present in the plant extract16.
Pheophorbides are one of several groups of compounds were isolated and characterized from Typhonium. This bioactive compound was investigated for anticancer properties, and results indicated the promising growth inhibitory effect and anticancer activities towards several cancer cells, including breast cancer cell. The anticancer activity of pheophorbides appears to be mediated through the induction of apoptotic cell death. Pheophorbides further decraled to exhibit other pharmacological efficacies, including antioxidant, antiinflammatory, antiallergic, antiviral, hepatoprotective and neuroprotective properties17. Based on previous investigation, T. flagelliforme had an bioactive compounds were identified as hexadecanoic acid methyl esters and octadecanoic acid (stearic acid)18. As shown in beforehand investigation, hexadecanoic acid methyl esters has been found to have antioxidant activity19, indicated that hexadecanoic acid methyl esters has potential to inhibit the growth of cancer cells. According to the previous report, hexadecanoic acid methyl esters capable to induce apoptosis and inhibit growth of human gastric cancer cells20, hexadecanoic acid (palmitic acid) also has a role induce apoptosis on cervical cancer cell lines21. Furthermore, hexadecanoic acid exhibited antitumor activity in mouse models and cytotoxic selective for MOLT-4 leukemia cancer cells in consequence of interaction with DNA topoisomerase I and ability to induce apoptosis22. Meanwhile, octadecanoic acid capable to inhibit proliferation development of human breast cancer cells in vitro23-25 and in vivo26 as delineated beforehand. The octadecanoic acid has also been reported to induce apoptosis in breast cancer cells and inhibit cell cycle of breast tumors27,28. Besides, epidemiological investigation have also presented that octadecanoic acid, has the potential to prevent and treat breast cancer29. In addition, study by Sianipar et al demonstrated that ethanolic extracts from rodent tuber (T. flagelliforme) mutant plants revealed cytotoxicity activity on breast cancer cells (MCF-7) performed by MTT assay method with IC50 values that were lower compared the control (mother plant)30.
CONCLUSION:
Rodent tuber (Typhonium flagelliforme (Lood) Bl) exhibited cancer chemopreventive activity against breast tumor. The T. flagelliforme extract had an ability to reduce the incidence of tumor significantly, suppressed the significant increase of tumor size, suppressed the amount and growth of tumors significantly, showed the different histopathologic figure and malignancy level towards the group given T. flagelliforme extract compared to the control group. This finding might emphasize the potency of T. flagelliforme as a cancer chemopreventive agent towards breast tumor from natural source.
ACKNOWLEDGEMENT:
This study was supported by Laboratory Grant Funds of Syiah Kuala University 2019 (Dana Hibah Laboratorium Universitas Syiah Kuala 2019).
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 31.10.2019 Modified on 13.02.2020
Accepted on 20.04.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(12):5811-5815.
DOI: 10.5958/0974-360X.2020.01013.6