Evaluation of Anti Cancer Activity of Kydia calycina Roxb. Leaf Extract on Different Cancer Cell Lines

 

Baburao Bhukya, Harikiran Lingabathula, Narsimhareddy Yellu*

Department of Pharmacology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India-506009

 

 

 

ABSTRACT:

To study the anticancer effects of Kydia calycina using various human cancer cell cultures, in vitro by MTT assay, ROS generation and caspase-3 activities. Human cervical carcinoma (HeLa) cells, human breast cancer (MCF-7) cells and human neuroblastoma (IMR-32) cells were maintained in a 5% CO₂ incubator at 37⁰C. Different concentrations of leaf extract of Kydia calycina in serum free culture medium were freshly prepared and used for cytotoxic activity by MTT assay, ROS generation and apoptotic effect by caspase-3 activity. The plant extract has revealed that greater percentage inhibition in all types of cancer cells in a dose dependent manner by MTT assay. The IC₅₀ values of K. calycina extract were found to be 41.17, 46.90 and 39.49 µg/mL against HeLa, MCF-7 and IMR-32 respectively. The apoptotic activity of Kydia calycina methanolic leaf extract was evaluated through ROS generation and caspase-3 activity. The results showed that the extract has significantly increased the ROS production and caspase-3 levels in all the cell cultures in a dose dependent manner. Further studies are warranted to elucidate the anticancer activity of K. calycina.

 

 

 

INTRODUCTION:

Plants and their products for treatment of diseases have been used extensively by humans for many years^1,2. Medicinal plants have gained huge interests from researchers around the world because of their positive biological activity. Chemoprevention, a relatively new and promising strategy to prevent cancer, is defined as the use of natural dietary compounds and/or synthetic substances to block, inhibit, reverse, or retard the process of carcinogenesis. Various cancer chemopreventative agents can encourage apoptosis in premalignant and malignant cells in vivo and/or in vitro, which is conceivably another anticancer mechanism^3,4.

 

Many of publications showed the effect of many herbal plants in treatment of wide range of illnesses. Also several naturally produced herbal formulations are currently available for cancer patients. As most of chemotherapeutic agents were cytotoxic to normal cells and developed drug resistance^5,6. Therefore scientific consideration and test of traditionally used herbs for the treatment of different malignancies could be also considered as a very valuable source for new chemotherapeutic drugs^6,7.

 

 Kydia calycina is distributed in Himalayas from the Indus eastwards to Myanmar and in peninsular India from Maharastra and Madhya Pradesh, principally in mixed, moist, deciduous forests. The leaves are applied to relieve body pains, lumbago and arthritis, a poultice of the leaves is used to treat skin diseases⁸. K. calycina leaf and stem bark paste applied for ulcers and skin diseases⁹. The K. calycina seed oil was reported to contain cyclopropenoid fatty acid apart from normal fatty acids¹⁰ and Hibiscoquinone B, Hibiscone C, 8-formyl-2,7-dihydroxy-5-isopropyl-1-methoxy-3-methyl naphthalene were isolated from stem heartwood of Kydia calycina¹¹. The Kydia calycina methanolic leaf extract was reported to having Analgesic, Anti-inflammatory and Hepatoprotective activity^12,13. So, the present study evaluated the cytotoxic effects of methanolic extracts of K. calycina using various human cancer cell cultures, in vitro.

 

MATERIALS AND METHODS:

Plant material:

The leaves of Kydia calycina were collected from Thirupathi hills, Andhara Pradesh, India. It was authenticated by Prof. V. Raju, Department of Botany, Kakatiya University, Warangal, India.

 

Preparation of extracts:

Kydia calycina leaf is were made free from the adherent foreign material and air-dried. Then they were coarsely powdered and macerated with methanol in a round bottom flask for 7 days separately. The content of the flask were stirred intermittently to ensure the efficiency of the extraction. After a week, they were filtered and concentrated under reduced pressure to yield corresponding extracts, and the extracts were kept in a desiccator to remove moisture and stored properly until used.

 

Cell culture and treatment:

Human cervical carcinoma (HeLa) cells, human breast cancer (MCF-7) cells and human neuroblastoma (IMR-32) cells were procured from NCCS, Pune. The cells were used between passages 10 and 15. All these cells were grown in suitable culture media supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin antibiotic solution. Cells were seeded at 250,000 cells/flask in a total volume of 10 mL.

 

When confluent, all the cells were trypsinized as described above, and seeded in 96 well plates at the rate of 1.0 x 10⁴ cells/0.1 mL. All the cell cultures were maintained in a 5% CO₂ incubator at 37⁰C. Different concentrations of extract of Kydia calycina such as in serum free culture medium were freshly prepared and used for cytotoxic activity.

 

MTT assay method:

Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population’s response to external factors. The reduction of tetrazolium salts is widely believed as a reliable way to study cell proliferation. The effect of test fractions on the cellular proliferation and viability was determined by using MTT assay method¹⁴. The yellow tetrazolium salt was reduced by dehydrogenase enzymes present in metabolically active cells, to produce reducing equivalents such as NADH and NADPH. The formazan product has low aqueous solubility and was present as purple crystals. The resulting formazan was dissolved by using dimethyl sulfoxide (DMSO) permitted the convenient quantification of product development. The intensity of the product color was measured at 562 nm and was directly proportional to the number of living cells in the culture¹⁵.

 

The adherent cells were trypsinized according to protocol and were re-suspended in fresh medium after centrifugation. Cell suspension was mixed thoroughly by pipetting several times to get a uniform single cell suspension. 10-15 passages were conducted before performing the experiment to evaluate the cytotoxicity. Different dilutions of fractions were made in media with final PBS+1% PEG (solvent control) concentration in the well to be less than 1%. 100 μL (0.1 mL) of cell suspension was transferred aseptically to each well of a 96 well plate and to it 100 μL of solvent/fraction (in triplicate) in media was added. The plate was then incubated at 37⁰C for 48 h in 5% CO₂ incubator. After 48 h of incubation, 20 μL of MTT was added to each well and the micro titer plate was again incubated for 2 h. 80 μL of lysis buffer was added to each well, the plate was wrapped in aluminum foil to prevent the oxidation of the dye. The plate was placed on a rotary shaker for 2 h to solubilize the purple formazan crystals. The absorbances were recorded on the ELISA reader at 562 nm wavelength. The absorbance of the test was compared with that of solvent control to get the percent cytotoxicity^16,17.

 

Measurement of ROS generation:

Generation of ROS was assessed by using a cell-permeable fluorescent signal H2DCF-DA as an indicator for ROS^18,19. As described previously, H2DCF-DA is oxidized to a highly green fluorescent 2070-dichlorofluorescein (DCF) by the generation of ROS. Cancer cell lines were pretreated with various concentrations of test extract for 24 h. After 24 h incubation period, the cells were washed with cold phosphate buffer solution (PBS) and incubated with 100 mmol/L H2DCF-DA for another 30 min at 37⁰C.

 

DCF fluorescence intensity was measured using the fluorescence plate reader (Varioskan Flash Multimode Reader, Waltham, MA) at excitation/emission of 488/525 nm. The determinations were carried out thrice in triplicate, ensuring each time that the number of cells per treatment group were the same to ensure reproducibility. The values were expressed as % relative fluorescence compared to the control.

Caspase-3 assay:

Caspases are members of the aspartate specific cysteinyl protease family. Caspase-3 exists in cells as an inactive 32 kDa proenzyme, called pro-caspase-3. Pro-caspase-3 is cleaved into active 17 and 12 kDa subunits by upstream proteases such as caspase-6, caspase-8 and granzyme B during apoptosis. The over expression of caspase-3 can result in initiation of apoptosis. Likewise, the inhibition of caspase-3 can prevent cells from entering into the apoptotic pathway. The activation of caspase-3 is used as a biomarker in evaluation of apoptosis and in understanding mechanisms of apoptosis induction²⁰.

 

The assay is based on colorimetric finding of the chromophore p-nitroaniline (pNA) after cleavage from the DEVD-pNA, a labeled substrate. The pNA light emission can be quantified using a microtiter plate reader at 405 nm. Assessment of the absorbances of pNA from an apoptotic sample with an uninduced control allows determination of the fold raise in caspase-3 activity²¹.

 

Statistical analysis:

Data were expressed as Mean±SEM. Significance was calculated using one way ANOVA followed by Dunnet’s multiple comparison tests compared to control.

 

RESULTS AND DISCUSSION:

Natural products or their derivatives have been exhibited to have significant anticancer potentials due to their ability to inhibit tumor growth, angiogenesis and metastasis without many side effects²². Recent publications showed the effect of many herbal plants in treatment of wide range of illnesses. Also several naturally produced herbal formulations are currently available for cancer patients. Most of the chemotherapeutic agents were cytotoxic to normal cells and build up drug resistance. Therefore scientific consideration and test of traditionally used herbs for the treatment of various malignancies could be also considered as a very important source for new chemotherapeutic drugs²³.

 

The in vitro cytotoxicity of methanolic extract of Kydia calycina were evaluated against three different cancer cell cultures, such as human cervical carcinoma (HeLa), human breast cancer (MCF-7) and human neuroblastoma (IMR-32) cells by using MTT assay, which is based on the reduction of MTT at different concentrations (10, 20, 40, 80 and 160 μg/ml). After 48 h of treatment, K calycina methanolic extract exhibited higher inhibitory effect against all tumor cells, with varying efficiencies and selectivities while others caused marginal cell inhibition (Figure 1). The IC₅₀ (concentration of the fraction causing 50% cell death) values of test fractions were given in Table 1. Among the methanolic extract have revealed that greater percentage inhibition in all types of cancer cells in a dose dependent manner. The IC₅₀ values of Kydia calycina extract were found to be 41.17, 46.90 and 39.49 µg/mL against HeLa, MCF-7 and IMR-32 respectively.

 

Figure 1. Effect of methanolic extract on cytotoxicity of HeLa, MCF-7 and IMR-32 cancer cell lines; Data were Mean±SEM

Table 1. The IC₅₀ values of different Kydia calcycina extracts using various cell lines

KM-Kydia calcycina Methanolic extract; STD- Cisplatin; Data were Mean±SEM; *p<0.05, **p<0.01 and ***p<0.001 compared to control.

 

Since the cytotoxicity of methanolic were greater, we further evaluated to reveal the mechanism for its cytotoxicity. For this purpose, we evaluated the apoptotic activity through ROS generation and caspase-3 activities of test extract all three cancer cell lines. The results showed that the both fractions have significantly increased the ROS production in all the cell cultures (Figure 2). Excess accumulation of ROS leads to cellular damage and inflammation of the tissues²⁴. ROS plays a major role in cellular senescence paving way to cell death; therefore there is an urgent need for potential therapeutics that may prevent oxidative stress induced neurodegeneration. It is highly possible that the pro-oxidant effect is responsible for the apoptotic activity of these extracts and ROS are key signaling molecules to modulate cell death²⁵. Accumulating evidence indicates that cancer cells produce high levels of ROS that lead to a state of increased basal oxidative stress. The increased production of ROS in cancer cells was observed in in vitro studies²⁶. Ahamad et al. demonstrated that naringenin leads to cell death in cancer cells via inducing ROS generation²⁷. We therefore investigated the effectiveness of test extracts in generation of ROS. We found that exposure of all three cancer cells with these extract dramatically enhanced generation of intracellular ROS at different levels in a dose dependent manner in all cell lines.

 

Figure 2. ROS generation effect of methanolic extract on HeLa, MCF-7 and IMR-32 cancer cell lines; Data were Mean±SEM

 

Intracellular caspase 3 activation is a key stage in the apoptotic pathway. Hence, we tested the effect of treatment with our plant fractions on intracellular caspase 3 enzymatic activity. Different human cancer cells were treated with the plant fractions and caspase 3 enzymatic activity within the cells was measured (Figure 3). Since caspase 3 activity rises while cells die and cell numbers drop, it was essential to normalize caspase 3 activity to the number of cells, to obtain more accurate results. The results here are expressed as the percent increase in caspase 3 activity in treated cells compared to cells added with DMSO. Caspase-3 levels were significantly increased with K calycina extract in HeLa (p<0.05), MCF-7 (p<0.05) and IMR-32 (p<0.01) cancer cells. Realizing the potential of plant derived compounds to develop as drugs against cancer, information on novel anti-cancer compounds from Himalayan region were retrieved from literature. The chemical structures of fifteen such anti-cancer compounds were modeled and optimized and their binding patterns were explored against nine selected molecular targets implicated in cell proliferation and apoptosis, which provided insights into their molecular mechanism of inhibition²⁸.

 

Figure 3. Caspase-3 levels of methanolic extract on HeLa, MCF-7 and IMR-32 cancer cell lines; Data were Mean±SEM

 

CONCLUSIONS:

In summary, among the four K. calycina leaf extract the showed increased percentage inhibition of MCF-7, HeLa and IMR-32 cells by MTT assay. Moreover, the apoptotic activity revealed that the plant extacts have displayed significant increased generation of ROS and caspase-3 activities in all cancer cell lines in a dose dependent fashion. Further studies are warranted to elucidate the molecular mechanisms of isolated compounds of Kydia calycina.

 

ACKNOWLEDGEMENTS:

The first author is thankful to University Grants Commission (UGC), New Delhi, INDIA for providing Post Doctoral Fellowship.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 11.07.2017       Modified on 19.08.2017

Accepted on 20.09.2017       ©A&V Publications All right reserved