Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line

Efeito apoptótico da natalensia bulbina e do clorofito comoso na linhagem celular mielogênea da leucemia K562

B. Padayachee F. Odun-Ayo L. Reddy About the authors

Abstract

Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Keyword:
Bulbine natalensis; Chlorophytum comosum; leukemia K562; apoptosis; cytotoxicity

Resumo

Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 μg / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p <0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

Palavras-chave:
Bulbine natalensis; Chlorophytum comosum; leucemia K562; apoptose; citotoxidade

1. Introduction

Cancer is a major health burden and the second leading cause of global death accounting for an estimated 8.2 million deaths annually (Stewart and Wild, 2014STEWART, B. and WILD, C., 2014. World cancer report 2014. Lyon, France: International Agency for Research on Cancer, World Health Organization, vol. 630.). The mortality rates of cancer are expected to increase in developing countries (Made et al., 2017MADE, F., WILSON, K., JINA, R., TLOTLENG, N., JACK, S., NTLEBI, V. and KOOTBODIEN, T., 2017. Distribution of cancer mortality rates by province in South Africa. Cancer Epidemiology, vol. 51, pp. 56-61. http://dx.doi.org/10.1016/j.canep.2017.10.007. PMid:29040965.
http://dx.doi.org/10.1016/j.canep.2017.1...
). Chronic myeloid leukaemia (CML) is a malignant blood cancer disease that affects the bone marrow cells characterized by the presence of an active tyrosine kinase (Apperley, 2015APPERLEY, J.F., 2015. Chronic myeloid leukaemia. Lancet, vol. 385, no. 9976, pp. 1447-1459. http://dx.doi.org/10.1016/S0140-6736(13)62120-0. PMid:25484026.
http://dx.doi.org/10.1016/S0140-6736(13)...
). Although, data on incidence and prevalence of CML in South Africa is lacking and limited as disease is often combined with the general category of leukaemia or other cancers (Stefan, 2015STEFAN, D.C., 2015. Why is cancer not a priority in South Africa? SAMJ: South African Medical Journal, vol. 105, no. 2, pp. 103-104. http://dx.doi.org/10.7196/SAMJ.9301. PMid:26242525.
http://dx.doi.org/10.7196/SAMJ.9301...
; Torre et al., 2015TORRE, L.A., BRAY, F., SIEGEL, R.L., FERLAY, J., LORTET-TIEULENT, J. and JEMAL, A., 2015. Global cancer statistics, 2012. CA: a Cancer Journal for Clinicians, vol. 65, no. 2, pp. 87-108. http://dx.doi.org/10.3322/caac.21262. PMid:25651787.
http://dx.doi.org/10.3322/caac.21262...
), the tendency of increased cases on a yearly basis is very high (Statistics South Africa, 2014STATISTICS SOUTH AFRICA, 2014. Mortality and causes of death in South Africa, 2013: findings from death notification. Pretoria: Statistics South Africa.). It has been projected there will be an estimate of 181,000 CML patients in the USA from 2050 (Huang et al., 2012HUANG, X., CORTES, J. and KANTARJIAN, H., 2012. Estimations of the increasing prevalence and plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy. Cancer, vol. 118, no. 12, pp. 3123-3127. http://dx.doi.org/10.1002/cncr.26679. PMid:22294282.
http://dx.doi.org/10.1002/cncr.26679...
). Certain anti-cancer drugs that are tyrosine kinase inhibitors are often the first line of treatment employed. However, they often cause adverse reactions, drug resistance and active recurrence of the leukaemia upon cessation of drug (Huang et al., 2012HUANG, X., CORTES, J. and KANTARJIAN, H., 2012. Estimations of the increasing prevalence and plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy. Cancer, vol. 118, no. 12, pp. 3123-3127. http://dx.doi.org/10.1002/cncr.26679. PMid:22294282.
http://dx.doi.org/10.1002/cncr.26679...
; O'Hare and Deininger, 2008O’HARE, T. and DEININGER, M.W., 2008. Toward a cure for chronic myeloid leukemia. Clinical Cancer Research, vol. 14, no. 24, pp. 7971-7974. http://dx.doi.org/10.1158/1078-0432.CCR-08-1486. PMid:19088011.
http://dx.doi.org/10.1158/1078-0432.CCR-...
). Hence, the development of an effective natural anti-cancer drug as an alternative medicine to treat this disease is required as a way to combat the increasing prevalence of the cancer.

Bulbine natalensis is a member of the family Asphodelaceae and a predominant species in the genus (Van Wyk and Gericke, 2000VAN WYK, B.-E. and GERICKE, N., 2000. People’s plants: a guide to useful plants of Southern Africa. África do Sul: Briza Publications.). This bulb-like indigenous soft plant is widely distributed in the northern and eastern province of South Africa. It is commonly known as Natal Bulbinella with a local name Ibhucu in Zulu and rooiwortel in afrikaans (Hutchings, 1996HUTCHINGS, A., 1996. Zulu medicinal plants: an inventory. South Africa: University of Natal Press.; Pather et al., 2011PATHER, N., VILJOEN, A.M. and KRAMER, B., 2011. A biochemical comparison of the in vivo effects of Bulbine frutescens and Bulbine natalensis on cutaneous wound healing. Journal of Ethnopharmacology, vol. 133, no. 2, pp. 364-370. http://dx.doi.org/10.1016/j.jep.2010.10.007. PMid:20937372.
http://dx.doi.org/10.1016/j.jep.2010.10....
). B. natalensis has found considerable attention for traditional medicinal applications for the treatment of skin diseases and wounds (Pather, 2009PATHER, N., 2009. The in vitro and in vivo effects of Bulbine frutescens and Bulbine natalensis on cutaneous wound healing. Braamfontein: University of the Witwatersrand, 223 p. Doctor of Philosophy Health Sciences.; Pather et al., 2011PATHER, N., VILJOEN, A.M. and KRAMER, B., 2011. A biochemical comparison of the in vivo effects of Bulbine frutescens and Bulbine natalensis on cutaneous wound healing. Journal of Ethnopharmacology, vol. 133, no. 2, pp. 364-370. http://dx.doi.org/10.1016/j.jep.2010.10.007. PMid:20937372.
http://dx.doi.org/10.1016/j.jep.2010.10....
), sexual dysfunction (Ajao et al., 2019AJAO, A., SIBIYA, N. and MOTEETEE, A., 2019. Sexual prowess from nature: a systematic review of medicinal plants used as aphrodisiacs and sexual dysfunction in sub-Saharan Africa. South African Journal of Botany, vol. 122, pp. 342-359. http://dx.doi.org/10.1016/j.sajb.2018.08.011.
http://dx.doi.org/10.1016/j.sajb.2018.08...
; Yakubu and Afolayan, 2009YAKUBU, M. and AFOLAYAN, A., 2009. Effect of aqueous extract of Bulbine natalensis (Baker) stem on the sexual behaviour of male rats. International Journal of Andrology, vol. 32, no. 6, pp. 629-636. http://dx.doi.org/10.1111/j.1365-2605.2008.00910.x. PMid:18710410.
http://dx.doi.org/10.1111/j.1365-2605.20...
) urinary infections and human immunodeficiency virus infection (Plessis-Stoman et al., 2009PLESSIS-STOMAN, D.D., DOWNING, T.G., VAN DE VENTER, M., and GOVENDER, S., 2009. Traditional herbal medicines: potential degradation of sterols and sterolins by microbial contaminants. South African Journal of Science, vol. 105, no. 3-4, pp. 147-150.). Chorophytum comosum is a grass-like evergreen perennial of the family Liliaceae and native to South Africa. C. comosum is widely distributed ranging from Sierra Leone to Ethiopia. However, it is a native plant from South Africa and has been found to be used in folk medicine to treat a range of ailments such as bronchitis, fractures and burn (Matsushita et al., 2005MATSUSHITA, H., KUWABARA, H., ISHIKAWA, S. and MOCHIZUKI, M., 2005. Apoptosis induced in human cell lines by a butanol extract from Chlorophytum comosum roots. Journal of Health Science, vol. 51, no. 3, pp. 341-345. http://dx.doi.org/10.1248/jhs.51.341.
http://dx.doi.org/10.1248/jhs.51.341...
) Previous reports have shown the protective effect of C. comosum against liver damage in rodent model (Areshidze et al., 2016ARESHIDZE, D., TIMCHENKO, L., GULYUKIN, M., KOZLOVA, M., RZHEPAKOVSKY, I., SEMIN, I. and ANDRAICHUK, V., 2016. Hepatoprotective effect of preparations produced from chlorophytum comosum (l.) at experimental toxic damage in wistar rats. Pharmacologyonline, vol. 2, pp. 81.) and potential to stimulate intestinal microbiome (Bondareva et al., 2017BONDAREVA, N., TIMCHENKO, L., DOBRYNYA, Y.M., AVANESYAN, S., PISKOV, S., RZHEPAKOVSKY, I., KOZLOVA, M., ARESHIDZE, D. and LYHVAR, A., 2017. Influence of the Chlorophytum comosum leaves hydroalcoholic extract on some representatives of intestinal microflora of rats. Journal of Pharmaceutical Sciences and Research, vol. 9, no. 6, pp. 874.).

Few studies have reported B. natalensis and C. comosum to be a potential major source for the treatment of cancers through its apoptotic roles and cytotoxic effect against some cancer cell lines including laryngeal carcinoma (HEp-2), human breast cancer (MDA-MB-231 and T47D), lung carcinoma (A549), human embryonic kidney 293 (HEK293), cervical carcinoma (HeLa), human promyelocytic leukaemia (HL-60) and pro-monocytic leukaemia (U937) (Kasumbwe et al., 2017KASUMBWE, K., VENUGOPALA, K.N., MOHANLALL, V., and ODHAV, B., 2017. Synthetic mono/di-halogenated coumarin derivatives and their anticancer properties. Anti-Cancer Agents in Medicinal Chemistry, vol. 17, no. 2, pp. 276-285. PMID: 27671300. http://dx.doi.org/10.2174/1871520616666160926112508.
http://dx.doi.org/10.2174/18715206166661...
; Kushwaha et al., 2019KUSHWAHA, P.P., VARDHAN, P.S., KAPEWANGOLO, P., SHUAIB, M., PRAJAPATI, S.K., SINGH, A.K. and KUMAR, S., 2019. Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells. Life Sciences, vol. 234, pp. 116783. http://dx.doi.org/10.1016/j.lfs.2019.116783. PMid:31442552.
http://dx.doi.org/10.1016/j.lfs.2019.116...
; Matsushita et al., 2005MATSUSHITA, H., KUWABARA, H., ISHIKAWA, S. and MOCHIZUKI, M., 2005. Apoptosis induced in human cell lines by a butanol extract from Chlorophytum comosum roots. Journal of Health Science, vol. 51, no. 3, pp. 341-345. http://dx.doi.org/10.1248/jhs.51.341.
http://dx.doi.org/10.1248/jhs.51.341...
; Singh and Reddy, 2012SINGH, R. and REDDY, L., 2012. Apoptosis in the human laryngeal carcinoma (HEp-2) cell line by bulbine natalensis and B frutescens fractions. International Journal of Biological & Pharmaceutical Research, vol. 3, no. 7, pp. 862-874.). However, no study has been reported, to the best of our knowledge, on the anti-cancer effect of B. natalensis and C. comosum on chronic human myelogenous leukaemia (K562) cell line. Hence, we investigated the apoptotic effect of B. natalensis and C. comosum organic and aqueous extracts through cytotoxicity, Hoechst bis-benzimide nuclear and flow cytometry annexin V staining of the K562 cells.

2. Materials and Methods

2.1. Plant material and extraction

Bulbine natalensis and Chorophytum comosum were obtained from the Reservoir Hills, Durban. The leaves, roots and bulbs of the plants were collected, washed with distilled water and air-dried for 48 h. The plant materials were blended to a fine powder and then stored. 10g of the powdered samples were added to 200 ml of sterile distilled water for the aqueous extracts and 100 ml methanol for the organic extracts. The plant material solvents were shaken at 156 rpm in an incubator of 37°C for 48 h. The leaf, root and bulb methanolic extracts for B. natalensis (BLM, BRM and BBM respectively) and leaf and root methanolic extracts for C. comosum (CLM and CRM respectively) were filtered through a 40µm filter disk (Whatman No 1, UK). The methanolic extracts were concentrated in a rotary evaporator at 65°C and subsequently stored in the biofreezer at -70°C. The root aqueous extract for B. natalensis (BRA) and leaf and root aqueous extracts for C. comosum (CLA and CRA respectively) were freeze dried for 24 h and then stored.

2.2. Cell lines

A human myelogenous (erythromyeloid) leukaemia K562 cell line was purchased from Highveld Biological, South Africa. These cells were non-adherent, rounded and resemble undifferentiated granulocytes. The cells were maintained with growth medium containing 10% foetal bovine serum (FBS) supplemented with 1% antibiotic (penicillin: 10,000 µ/ml, streptomycin sulphate: 10,000 µ/ml) in a humidified 5% CO2 air incubator at 37°C. Cultures were examined daily by the morphological observation, colour of medium, cells density and growth pattern using Zeiss inverted light microscope. Cells were washed and preserved in medium containing 90% FBS and 10% (100 μl) dimethyl sulfoxide (DMSO) at -70°C.

2.3. Determination of cytotoxicity effect of B. natalensis and C. comosum on K562 cells using MTT Assay

The cytotoxicity effect of B. natalensis and C. comosum methanolic and aqueous extracts were quantified using the MTT assay following the method by (Jun et al., 2003JUN, D.Y., RUE, S.W., HAN, K.H., TAUB, D., LEE, Y.S., BAE, Y.S. and KIM, Y.H., 2003. Mechanism underlying cytotoxicity of thialysine, lysine analog, toward human acute leukemia Jurkat T cells. Biochemical Pharmacology, vol. 66, no. 12, pp. 2291-2300. http://dx.doi.org/10.1016/j.bcp.2003.08.030. PMid:14637187.
http://dx.doi.org/10.1016/j.bcp.2003.08....
). The percentage of cytotoxicity (%) were calculated using Equation 1 as described below (Singh and Reddy, 2012).SINGH, R. and REDDY, L., 2012. Apoptosis in the human laryngeal carcinoma (HEp-2) cell line by bulbine natalensis and B frutescens fractions. International Journal of Biological & Pharmaceutical Research, vol. 3, no. 7, pp. 862-874. Briefly, the K562 cells (8×104 cells/ml) were seeded in a 96-well microtiter plate (100 μl/well) and subsequently treated with the B. natalensis and C. comosum methanolic and aqueous extracts at 100 and 1,000 μg/ml concentrations for 24 h. Untreated cultured wells containing the K562 cells with complete culture medium and 2% DMSO were used as the control. All samples were assayed in triplicate. The plate was centrifuged at 400 g for 10 min and the cell supernatant was removed. MTT solution (10 μl) was added to each well and incubated for 3 h at 37°C humid chamber. The purple formazan crystals were solubilized with 100 μl DMSO and incubated further for 30 min. The absorbance was read at 570 nm with a reference wavelength of 650 nm using a microplate reader (BioTek Instruments, Inc. USA). The cytotoxicity (%) was calculated:

Cytotoxicity%=100-Average absorbance of treated cellsAverage absorbance of control cells×100(1) (Singh and Reddy, 2012)

2.4. Determination of DNA fragmentation and condensation in K562 cells

The Hoechst bis-benzimide (H33342), a blue-fluorescent dye that binds to the minor groves of DNA determines the condensed pycnotic nuclei in apoptotic cells. Briefly, 300 μl of 5 x 104 cells/ml in 24-well microtiter plate treated with 6 μl of the B. natalensis and C. comosum methanolic and aqueous extracts at 37°C were incubated for 24 - 48 h. Positive control cells were treated with 10 μg/ml camptothecin while untreated cells remains as negative control. Following incubation, after cells were centrifuged at 1500 rpm for 5 min, the cell pellets were washed with 500 μl of 0.1M PBS and centrifuged again as previously described. H33342 solution (100 μl) was added and incubated for 15 min at 37°C. The cell pellets were retrieved and washed as previously described. The cells were fixed in 10% paraformaldehyde solution for 5 min, centrifuged and washed. Finally, cells were re-suspended in 100 μl of PBS, wet mounted on coverslips and observed immediately under the Zeiss AxioPlan fluorescence (Zeiss, Germany) microscope with excitation filter of 350 nm and barrier filter of 450 nm.

2.5. Flow cytometry apoptotic cell death analysis by annexin V staining

The induced apoptotic cell death population by the methanolic and aqueous extracts of B. natalensis and C. comosum in K562 cells was determined using flow cytometry technique as previously described (Wlodkowic et al., 2009WLODKOWIC, D., SKOMMER, J. and DARZYNKIEWICZ, Z., 2009. Flow cytometry-based apoptosis detection. Methods in Molecular Biology (Clifton, N.J.), vol. 559, pp. 19-32. PMid:19609746.). A 400 μl of 2 x 105 cells/ml were incubated in T252 flasks for 24 h at 37°C. Cells were treated with 8 μl of the methanolic and aqueous extracts of B. natalensis and C. comosum and incubated further for 24 h at 37°C. Cells were washed in cold PBS and centrifuged at 1,500 rpm for 10 min. Following another washing, cells were re-suspended in a prepared 1X annexin binding buffer which contains 1 ml of 5X annexin binding buffer added to 4 ml of de-ionized water. Alexa fluor 488 annexin V (5 μl) and SYTOX green working solution (1 μl) were added to the cells and incubated at room temperature for 15 min. Furthermore, 400 μl of 1X annexin binding buffer was added to the cells, mixed gently while kept on ice and stained cells were immediately analysed by flow cytometry measuring the fluorescence emission at 530 nm.

2.6. Statistical analysis

All data were presented as the mean ± standard deviation at triplicate independent experiments. All statistical data were analysed using GraphPad Prism version 5.01 software. Statistical comparisons between the treatments extracts were performed using t-test and one-way analysis of variance. P < 0.0001 was considered to indicate a statistically significant difference.

3. Result and Discussion

This present in vitro study utilised the leaf, root, and bulb methanolic and aqueous extracts of both B. natalensis and C. comosum to assess the apoptotic effect of the two plants in myelogenous leukaemia K562 cell line. Few studies have reported anti-cancer properties of B. natalensis in vitro and chemoprotective properties of Cholophytum comosum in a liver damaged in vivo model (Areshidze et al., 2016ARESHIDZE, D., TIMCHENKO, L., GULYUKIN, M., KOZLOVA, M., RZHEPAKOVSKY, I., SEMIN, I. and ANDRAICHUK, V., 2016. Hepatoprotective effect of preparations produced from chlorophytum comosum (l.) at experimental toxic damage in wistar rats. Pharmacologyonline, vol. 2, pp. 81.; Singh and Reddy, 2012SINGH, R. and REDDY, L., 2012. Apoptosis in the human laryngeal carcinoma (HEp-2) cell line by bulbine natalensis and B frutescens fractions. International Journal of Biological & Pharmaceutical Research, vol. 3, no. 7, pp. 862-874.). Potential metabolites in B. natalensis and C. comosum has been shown to inhibit the growth of some cancer cells lines. However, there is little or no report known till date about the effects of these two medicinal plants on K562 cells, a type of cell line originated from a CML patient with hematopoietic stem cells disorder.

In this present study, the dose-dependent cytotoxicity effect of the methanolic and aqueous extracts of B. natalensis and C. comosum on the viability of K562 cells was observed (See Figure 1A). A high cytotoxicity activity was exhibited by the methanolic and aqueous extracts of C. comosum. The CRM extract showed a high cytotoxicity (16.7%) against the K562 cell line at 1,000 μg/ml concentration (See Figure 1B). An increase in cytotoxicity was exhibited from 4.9% at 100 μg/ml to 8.6% at 1,000 μg/ml concentration in the K562 cell by the BRM extract. The root methanolic extracts of both plants showed significant cytotoxicity (p < 0.0001) against the K562 cell line compared to the leaf, bulb methanolic and root aqueous extracts irrespective of the concentration. This suggests the cytotoxic efficacy of the root methanolic extracts of B. natalensis and C. comosum against K562 cell proliferation. Conversely, the leaf methanolic (BLM and CLM) extracts of both plants extracts tend to show the lowest cytotoxicity irrespective of the concentration.

Figure 1
Cytotoxic effect of (A) Bulbine natalensis and (B) Cholophytum comosum extracts at 100 μg/ml and 1,000 μg/ml concentrations in human myelogenous leukaemia K562 cell line using MTT assay. All *** is p < 0.0001 which is considered statistically significant.

The extract treatments of B. natalensis and C. comosum showed cytotoxic effect to the K562 cells in a concentration-dependent manner. We observed a high cytotoxicity in the C. comosum extracts as the root methanolic extract was higher compared to the leaf methanolic and aqueous extracts. Matsushita et al. (2005)MATSUSHITA, H., KUWABARA, H., ISHIKAWA, S. and MOCHIZUKI, M., 2005. Apoptosis induced in human cell lines by a butanol extract from Chlorophytum comosum roots. Journal of Health Science, vol. 51, no. 3, pp. 341-345. http://dx.doi.org/10.1248/jhs.51.341.
http://dx.doi.org/10.1248/jhs.51.341...
, showed that root butanol extract of C. comosum induces anti-proliferative effect in HL-60 and U937 cell lines However, Adhami et al. (2020)ADHAMI, S., FAROOQI, H., ABDIN, M. Z., PRASAD, R., and MALIK, A. A., 2020. LC-ESI-MS/GC-MS based metabolite profiling of Chlorophytum comosum (Thunb.) Jaques and evaluation of its antioxidant and antiproliferative effects on lung and breast cancer cell lines. Research Square, pp. 1-38. http://dx.doi.org/10.21203/rs.3.rs-29686/v1.
http://dx.doi.org/10.21203/rs.3.rs-29686...
reported the leaf ethanolic extract of C. comosum exhibited maximum response of cytotoxicity (IC50 3.08 μg/ml) compared to the root extract. A systematically screening of the cytotoxicity activity of the different phytochemical extracts from the plant extracts may provide a valuable information to this controversy. In addition, we noticed that the root extract was more cytotoxic than the other methanolic and aqueous extracts of B. natalensis against the K562 cell line at 1,000 μg/ml. Although the cytotoxicity level of the CRM was significantly higher compared to the BRM, it is plausible to assume that the type and sensitivity of the phytochemical compounds such as anthraquinones, chrysophanol and knipholone present in the root has a potent anti-proliferative activity reacting in a different apoptotic pathway (Musara and Aladejana, 2020MUSARA, C. and ALADEJANA, E.B., 2020. Review of studies on Bulbine natalensis Baker (Asphodelaceae): ethnobotanical uses, biological and chemical properties. Journal of Applied Pharmaceutical Science, vol. 10, no. 9, pp. 150-155.) and thus may differently affect the proliferation of the K562 cell line.

The morphological and cellular changes in the K562 cell induced by the extracts of B. natalensis and C. comosum at 1,000 μg/ml concentration after staining with the H33342 fluorescent probe showed indicative apoptotic characteristic such as membrane damage, nuclear fragmentation, vacuolated cytoplasm and possible nuclei disintegration in comparison to the untreated cells (See Figure 2). These were indicative of apoptosis in the treated K562 cells which exhibited different degree of cell and nuclear fragmentation. The BLM, BRM, BBM, CLA and CRA extracts treated K562 cells showed possible morphological loss of cell membrane integrity causing membrane blebbing (MB) and fragmentation (See Figure 2A-2D, 2G, 2H). Degradation of the cell cytoplasm were observed in the BRM and CRM extracts treated K562 cells (see Figure 2F). Vacuolated cytoplasm (VC) was also presented in the CLM and CRA extracts treated K562 cells (see Figure 2E and 2J). This suggests that the root methanolic extracts of both plants is induced more apoptotic activity when compared to the other extracts in their aqueous forms. The K562 cells treated with CLA extracts at 100 μg/ml concentration induced little or no apoptotic activity as cellular disintegration was very minimal (See Figure 2I). The camptothecin treated K562 cells showed vacuolated condensed nuclei with extensive membrane damage (see Figure 2K) compared to the untreated cells exhibiting a normal nuclear morphology (see Figure 2L).

Figure 2
H33342 staining viewed under fluorescent microscope on human myelogenous leukaemia K562 cell morphological changes induced by: 1. 1,000 μg/ml Bulbine natalensis (A) leaf methanolic extract, BLM (B) root methanolic extract, BRM (C) bulb methanolic extract, BBM and (D) root aqueous extract, BRA; 2. 1,000 μg/ml Cholophytum comosum (E) leaf methanolic extract, CLM (F) root methanolic extract, CRM (G) leaf aqueous extract, CLA (H) root aqueous extract, CRA; 100 μg/ml Cholophytum comosum (I) leaf aqueous extract, CLA (J) root aqueous extract, CRA and 3. Controls (K) camptothecin indicating membrane blebbing (L) untreated cells indicating normal cells. MB- membrane blebbing; VC- vacuolated cytoplasm.

Kushwaha et al. (2019)KUSHWAHA, P.P., VARDHAN, P.S., KAPEWANGOLO, P., SHUAIB, M., PRAJAPATI, S.K., SINGH, A.K. and KUMAR, S., 2019. Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells. Life Sciences, vol. 234, pp. 116783. http://dx.doi.org/10.1016/j.lfs.2019.116783. PMid:31442552.
http://dx.doi.org/10.1016/j.lfs.2019.116...
reported a significant number of apoptotic cells were observed in the MDA-MB-231 breast cancer cells treated with leaf methanolic extracts of B. frutescens. The expression of caspase-3 induced by B. natalensis ethanol fractions extract in Hep-2 cells as against the aqueous extract was indicative of the apoptosis (Singh and Reddy, 2012SINGH, R. and REDDY, L., 2012. Apoptosis in the human laryngeal carcinoma (HEp-2) cell line by bulbine natalensis and B frutescens fractions. International Journal of Biological & Pharmaceutical Research, vol. 3, no. 7, pp. 862-874.). In the present study, the induced K562 cell morphological changes was more evident in the 1,000 μg/ml dose concentration. The different dose treatments applied to K562 cell could possibly induced different morphological alterations of the cellular surface which corroborate with Kushwaha et al. (2019)KUSHWAHA, P.P., VARDHAN, P.S., KAPEWANGOLO, P., SHUAIB, M., PRAJAPATI, S.K., SINGH, A.K. and KUMAR, S., 2019. Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells. Life Sciences, vol. 234, pp. 116783. http://dx.doi.org/10.1016/j.lfs.2019.116783. PMid:31442552.
http://dx.doi.org/10.1016/j.lfs.2019.116...
and Rahman et al. (2013)RAHMAN, S.N.S.A., WAHAB, N.A. and ABD MALEK, S.N., 2013. In vitro morphological assessment of apoptosis induced by antiproliferative constituents from the rhizomes of Curcuma zedoaria. Evidence-based Complementary and Alternative Medicine: eCAM, vol. 2013, pp. 257108. http://dx.doi.org/10.1155/2013/257108.
http://dx.doi.org/10.1155/2013/257108...
.

Comparatively, the better efficacy of C. comosum extracts against K562 cell in the cytotoxicity study motivate for further confirmation by annexin V staining through quantitative flow cytometry analysis to justify the apoptotic potential of both B. natalensis and C. comosum extracts in K562 cells. B. natalensis and C. comosum extracts treated K562 cells were stained with a recombinant Annexin V dye conjugated to an Alexa fluor 488 dye to quantify the apoptotic induction ability of the plants extract. Viable cells were stained Annexin V negative and apoptotic cells were Annexin V positive. In this study, C. comosum has a significantly higher efficiency of inducing apoptosis most especially the leaf methanolic and root aqueous extracts.

The extracts: BLM (85.1%), BRM (71.9%), BBM (80.6%), CLM (81.1%) and CRA (88.7%) induced a significantly high apoptosis (p < 0.0001) in the K562 cells compared to the BRA (40.9%), CRM (41.0%) and CLA (53.2%) at 1,000 μg/ml concentration. All B. natalensis extract treated K562 cells showed low apoptotic cell population level below 50% at 100 μg/ml concentration (see Figure 3A). Contrarily, it was observed that CLM (71.9%) and CRA (75.5%) extract treated K562 cells exhibited apoptosis significantly (p < 0.0001) high above 50% at 100 μg/ml concentration (see Figure 3B). Furthermore, a significantly high apoptosis (p < 0.0001) was induced in the K562 cells by both the leaf and root extracts of the C. comosum irrespective of the dose concentration compared to the B. natalensis extracts. This further confirms the high cytotoxicity observed in the C. comosum extract treated K562 cell. Although, previous studies suggested that a specific hydrolysate, DL – ornithine monohydrochloride, which is present in the aerial part of C. comosum may induce significant apoptotic through mitosis, necrotic and anti-proliferation effects (Areshidze et al., 2016ARESHIDZE, D., TIMCHENKO, L., GULYUKIN, M., KOZLOVA, M., RZHEPAKOVSKY, I., SEMIN, I. and ANDRAICHUK, V., 2016. Hepatoprotective effect of preparations produced from chlorophytum comosum (l.) at experimental toxic damage in wistar rats. Pharmacologyonline, vol. 2, pp. 81.; Areshidze et al., 2013ARESHIDZE, D.A., TIMCHENKO, L.D. and KOZLOVA, M.A., 2013. Protective effect of enzymatic hydrolyzate of Chlorophytum comosum (L.) against experimental toxic liver injury in Wistar rats at the age of two years. American Journal of Bioscience and Bioengineering, vol. 1, no. 4, pp. 49-54. http://dx.doi.org/10.11648/j.bio.20130104.11.
http://dx.doi.org/10.11648/j.bio.2013010...
). Our study agrees with Areshidze et al. (2016)ARESHIDZE, D., TIMCHENKO, L., GULYUKIN, M., KOZLOVA, M., RZHEPAKOVSKY, I., SEMIN, I. and ANDRAICHUK, V., 2016. Hepatoprotective effect of preparations produced from chlorophytum comosum (l.) at experimental toxic damage in wistar rats. Pharmacologyonline, vol. 2, pp. 81. considering the high cytotoxicity and apoptotic effect of C. comosum.

Figure 3
Apoptotic effect of (A) Bulbine natalensis and (B) Cholophytum comosum extracts at 100 μg/ml and 1,000 μg/ml concentrations in human myelogenous leukaemia K562 cell line using Annexin V. All *** is p < 0.0001 which is considered statistically significant.

4. Conclusion

In this study, the methanolic and aqueous extracts of B. natalensis and C. comosum inhibit the proliferation of K562 cells possibly at the early stage. It is plausible that B. natalensis and C. comosum possess apoptotic effect with considerable cytotoxicity against the in vitro myelogenous leukaemia. Previous studies have reported anti-proliferative property of plants extracts against HL-60, U937, HEp-2, MDA-MB-231 and T47D cancer cell lines in a time-dependent fashion within 24, 48 and 72 hr response to treatment (Kushwaha et al., 2019KUSHWAHA, P.P., VARDHAN, P.S., KAPEWANGOLO, P., SHUAIB, M., PRAJAPATI, S.K., SINGH, A.K. and KUMAR, S., 2019. Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells. Life Sciences, vol. 234, pp. 116783. http://dx.doi.org/10.1016/j.lfs.2019.116783. PMid:31442552.
http://dx.doi.org/10.1016/j.lfs.2019.116...
; Matsushita et al., 2005MATSUSHITA, H., KUWABARA, H., ISHIKAWA, S. and MOCHIZUKI, M., 2005. Apoptosis induced in human cell lines by a butanol extract from Chlorophytum comosum roots. Journal of Health Science, vol. 51, no. 3, pp. 341-345. http://dx.doi.org/10.1248/jhs.51.341.
http://dx.doi.org/10.1248/jhs.51.341...
). The limitation to this study is that the 48 – 72 hrs treatment of the B. natalensis and C. comosum extracts against the K562 cell line which may increase cytotoxicity against the growth of the cells. However, this warrant further studies to elucidate the time response effect of treatment and ascertain the apoptotic pathway induced by B. natalensis and C. comosum in both in vivo and preclinical models. This might contribute to the overall apoptotic properties of B. natalensis and C. comosum and to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Acknowledgements

We express our gratitude to the National Research Foundation for financial assistance to carry out this study.

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Publication Dates

  • Publication in this collection
    20 Dec 2021
  • Date of issue
    2024

History

  • Received
    20 Apr 2021
  • Accepted
    24 Aug 2021
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