Table of Contents    
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 2  |  Page : 201-206  

Cytotoxic activities of the dichloromethane extracts from Andrographis paniculata (Burm. f.) nees


1 Department of Chemistry, De La Salle University, Manila 1004, Philippines
2 Department of Biology, De La Salle University; Center for Natural Science and Environmental Research, De la Salle University, Manila 1004, Philippines
3 Division of Chinese Medicinal Chemistry, National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan
4 Department of Chemistry, De La Salle University, Manila 1004; Department of Chemistry, De La Salle University Science and Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines

Date of Web Publication20-Jun-2018

Correspondence Address:
Consolacion Y Ragasa
Department of Chemistry, De La Salle University, 2401 Taft Avenue, Manila 1004
Philippines
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_91_17

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   Abstract 

Introduction: Although diterpenes from Andrographis paniculata Burm.f. Nees have been found to have chemotherapeutic activity, a thorough investigation on the cytotoxic and anti-proliferative analyses on different cancer cell lines using these isolated constituents has not been achieved. Objectives: The primary objective of this study was to probe the cytotoxic capacity of the labdane diterpenoids andrographolide (1), 14-deoxyandrographolide (2), 14-deoxy-12-hydroxyandrographolide (3), and neoandrographolide (4) on mutant and wild type immortalized cell lines. Methods: Breast adenocarcinoma (MCF-7), colon carcinomas (HCT-116 and HT-29), small cell lung carcinoma (H69PR), human acute monocytic leukemia (THP-1), and wild type primary normal human dermal fibroblasts - neonatal cells (HDFn) were incubated with 1-4 and the degree of cytotoxicity was analyzed by employing the in vitro PrestoBlue® cell viability assay. Working solutions of 1-4 were prepared in complete cell culture medium to a final non-toxic DMSO concentration of 0.2%. The plates were incubated at 37°C with 5% CO2 in a 98% humidified incubator throughout the assay. Nonlinear regression and statistical analyses were done to extrapolate the half maximal inhibitory concentration, IC50. One-way ANOVA (P < 0.05) and multiple comparison, Tukey's post hoc test (P < 0.05), was used to compare different pairs of data sets. Results were considered significant at P < 0.05. Results: The highest cytotoxicity index was exhibited by the H69PR and 1 trials which displayed the lowest IC50 value of 3.66 μg/mL, followed by HT-29 treated with 2, HCT-116 and 1 trials, and H69PR treated with 4 (IC50 = 3.81, 3.82 and 4.19 μg/mL, respectively). Only 1 and 4 were detrimental towards MCF-7; while 1, 3, and 4 were degenerative against H69PR. Tukey's post hoc multiple comparison indicated no significant difference in the cytotoxicity of 1-4 on HCT-116 cells which afforded IC50 values ranging from 3.82 to 5.12 μg/mL. Evaluation of the two colon carcinoma cell lines showed that HCT-116 was categorically more susceptible to cellular damage caused by treatments with 1-4 than was HT-29. Cytotoxicity was not detected in THP-1 and HDFn cells (IC50 >100 μg/mL). Conclusion: Diterpenoids 1-4 isolated from the dichloromethane extract of the leaves of A. paniculata exhibited different cytotoxic activities against MCF-7, HCT-116, HT-29, H69PR. All constituents had comparable action on HCT-116 cells but were not found to be cytotoxic to normal HDFn cells and mutant THP-1 cells.

Keywords: Andrographis paniculata (Burm. f.) Nees, cytotoxicity, PrestoBlue cell viability assay


How to cite this article:
S. Tan MC, Oyong GG, Shen CC, Ragasa CY. Cytotoxic activities of the dichloromethane extracts from Andrographis paniculata (Burm. f.) nees. J Nat Sc Biol Med 2018;9:201-6

How to cite this URL:
S. Tan MC, Oyong GG, Shen CC, Ragasa CY. Cytotoxic activities of the dichloromethane extracts from Andrographis paniculata (Burm. f.) nees. J Nat Sc Biol Med [serial online] 2018 [cited 2018 Dec 12];9:201-6. Available from: http://www.jnsbm.org/text.asp?2018/9/2/201/234731


   Introduction Top


Andrographis paniculata (Burm. f.) Nees has been found to have anticancer and immunostimulatory constituents.[1] This medicinal herb, endemic to Taiwan, Mainland China, and India, imparts an unpleasant bitter taste and is used to remedy liver disorders, bowel complaints of children, colic pain, common cold, and upper respiratory tract infections.[2],[3],[4] The aerial part of A. paniculata is commonly used in Chinese medicine. A. paniculata's healing ability is achieved by its capacity to “cool” or to relieve internal heat, inflammation, and pain. It is also used for detoxification or cleansing.[5],[6],[7]

The bioactivity of A. paniculata is attributed to diterpenoids, flavonoids, and polyphenols.[8],[9] From the ethyl acetate soluble fraction of the ethanol or methanol extract, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7, 8, 2',5'-tetramethoxyflavone, 5-hydroxy-7, 8, 2',3'-tetramethoxyflavone, 5-hydroxy-7, 8, 2'-trimethoxyflavone, 7-O-methylwogonin, and 2'-methyl ether were isolated as the main flavonoids.[10],[11],[12] Andrographolide is the major diterpenoid in A. paniculata, making up about 4%, 0.8%–1.2%, and 0.5%–6% in dried whole plant, stem, and leaf extracts, respectively.[13],[14],[15] The other main diterpenoids are deoxyandrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and isoandrographolide.[11],[13]

We previously reported the chemical investigation of the dichloromethane extracts of A. paniculata which led to the isolation of andrographolide, 14-deoxyandrographolide, 14-deoxy-12-hydroxyandrographolide, a mixture of β-sitosterol and stigmasterol in a 3:1 ratio, and chlorophyll a from the leaves; a mixture of β-sitosterol and stigmasterol in a 3:2 ratio, 5,2'-dihydroxy-7,8-dimethoxyflavone or skullcapflavone I, and a mixture of long-chain trans-cinnamate esters and β-sitosteryl fatty acid esters from the roots; β-sitosterol, monogalactosyl diacylglycerols, lupeol, and triacylglycerols from the pods; and 14-deoxyandrographolide from the stems.[16] Further investigation of the A. paniculata dichloromethane extracts led to the isolation of squalene, polyprenol, lutein, chlorophyll a, and a mixture of β-sitosterol and stigmasterol in a 3:1 ratio from the stems; α-amyrin acetate, triacylglycerols, and a mixture of lupeol, α-amyrin, and β-amyrin in a 2:2:1 ratio from the leaves.[17] In our most recent work, the constituents identified were neoandrographolide, 1,5-dimethyl-1,5-cyclooctadiene and 2-hydroxyethyl benzoate, and squalene from the leaves while the stems yielded neoandrographolide and 1,5-dimethyl-1,5-cyclooctadiene.[18]

In this work, we established the cytotoxic activities of the crude dichloromethane extracts of the different parts of A. paniculata against H69PR, human breast adenocarcinoma cell line (MCF-7), acute monocytic leukemia (THP-1), human colorectal carcinoma cell line (HCT-116), and human small cell lung cancer cell line (HT-29). To the best of our knowledge, this is the first reported study using this methodology of cytotoxic and antiproliferative analyses on the crude dichloromethane extracts of the leaves, roots, stems, and pods of A. paniculata against the aforementioned human cancer cells.


   Materials and Methods Top


Sample collection

The Andrographis paniculata (Burm. f.) Nees leaves were collected from Abucay, Bataan, in September 2015. The plant was authenticated at the Botany Division, Philippine National Museum.

Preparation of extracts

Lyophilized plant parts leaves (3.33 g), roots (3.20 g), stems (4.64 g), and pods (3.18 g) were ground in an osterizer, incubated for 3 days in CH2 Cl2, then filtered. The filtrates were concentrated under vacuum to afford crude extracts of leaves (43.7 mg), roots (8.4 mg), stems (2.7 mg), and pods (9.1 mg). The crude extracts of A. paniculata were dissolved in dimethyl sulfoxide (DMSO) to make a 4 mg/mL stock solution. Working solutions were prepared in complete cell culture medium to a final nontoxic DMSO concentration of 0.2%.

Maintenance and preparation of cells

The bioactivity of the dichloromethane (CH2 Cl2) extracts from A. paniculata was tested on the following human cell lines (ATCC, Manassas, Virginia, USA.): breast cancer (MCF-7), colon cancer (HCT-116 and HT-29), small cell lung carcinoma (H69PR), human THP-1, and a primary culture of normal human dermal fibroblasts-neonatal (HDFn) (Thermo Fisher Scientific, Gibco ®, USA) which are routinely maintained at the Cell and Tissue Culture Laboratory, Molecular Science Unit, Center for Natural Sciences and Environmental Research, De La Salle University. Following standard procedures,[19] cells were grown in complete growth medium composed of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific Gibco®, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Gibco®, USA) and 1x antibiotic-antimycotic (Thermo Fisher Scientific, Gibco®, USA) and kept at 37°C with 5% CO2 in a 98% humidified incubator. Upon reaching 80% confluence, the monolayer cultures were washed with phosphate-buffered saline (pH 7.4, Thermo Fisher Scientific, Gibco®, USA), trypsinized with 0.05% trypsin-ethylenediaminetetraacetic acid (Thermo Fisher Scientific, Gibco®, USA) and resuspended with complete fresh media. Cells were counted following standard trypan blue exclusion method using 0.4% Trypan Blue Solution (Thermo Fisher Scientific, Gibco®, USA). Cells were later seeded in 100 μL aliquots into 96-well microtiter plates (Falcon , USA) with a final viable density of 1 × 104 cells/well. Uniform viable cell density is a crucial aspect to be considered when performing viability analysis. Resulting data from cell physiology and response to cytotoxic compounds are affected by the initial cell density; cells of lower densities are more disturbed by the toxins. Adversely, toxins may become bound and unavailable at higher cell densities.[20] It is for this reason why the researchers chose this level of cell density. The plates were further incubated overnight at 37°C with 5% CO2 in a 98% humidified incubator until complete cell attachment was achieved. These plates were used for the bioassay as described in the succeeding section.

Cell viability assay

The cytotoxic activities of the A. paniculata extracts were determined using PrestoBlue® (Thermo Fisher Scientific, Molecular Probes®, Invitrogen, USA) viability assay. The bioassay is based on the presence of mitochondrial reductases in viable cells which metabolically convert the resazurin dye (blue and nonfluorescent) to resorufin (red and highly fluorescent). The conversion is proportional to the number of metabolically active and viable cells and is relatively determined quantitatively using absorbance measurements at 570 nm. To the monolayers in the microtiter plate, 100 μL of filter-sterilized crude leaf, root, stem, and pod extracts were, respectively, added to corresponding wells at two-fold serial dilutions to make final screening concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 μg/mL. The treated cells were further incubated for 4 days at 37°C in 5% CO2 and 98% humidity. Twenty microliters of PrestoBlue® was added to each well. The cells were incubated for 1 h at 37°C in 5% CO2 and 98% humidity. Wells with no sample added served as negative controls and wells with Zeocin (Thermo Fisher Scientific, Gibco®, USA) served as the positive control. Zeocin, which is a selective antibiotic of the bleomycin/phleomycin family, can kill most cells growing aerobically from 0.5 to 1000 mg/mL. Absorbance measurements were carried out using BioTek ELx800 Absorbance Microplate Reader (BioTek® Instruments, Inc., USA) at 570 nm and normalized to 600 nm values (reference wavelength). Absorbance readings were used to calculate for the cell viability for each sample concentration following the equation below:



Nonlinear regression and statistical analyses were done using GraphPad Prism 7.01 (GraphPad Software, Inc., USA) to extrapolate the half maximal inhibitory concentration, IC50(the concentration of the compound which resulted in a 50% reduction in cell viability). The cytotoxicity of the crude leaf, root, stem, and pod extracts was expressed as IC50 values. All tests were performed in triplicates and data were shown as mean ± standard error of the mean (SEM). The extra sum-of-squares F-test or Brown–Forsythe test was used to evaluate the differences in the best-fit parameters (half maximal inhibitory concentration) among data sets (treatments) and to determine the differences among dose-response curve fits according to the software's recommended approach. One-way analysis of variance (ANOVA) (P< 0.05) was also conducted to determine significant differences among group variables, followed by the multiple comparisons, Tukey's post hoc test (P< 0.05), to compare different pairs of data sets. Results were considered significant at P < 0.05.


   Results and Discussion Top


The cytotoxicity of the plant parts of A. paniculata leaves, roots, stems, and pods was investigated on H69PR, MCF-7, THP-1, HT-29, HCT-116, THP-1, and HDFn. Analyses of the cytotoxicity of the leaf, root, and pod extracts on HDFn resulted to IC50 values exceeding 100 mg/mL while the stem extract exhibited an IC50 value of 72.22 mg/mL; Zeocin, an intercalating agent, was used as the positive control in all the trials. An illustration of the percent cell viability as a function of the logarithmic function of the concentrations used is presented in [Figure 1]. The characteristic inhibitory dose-response, which is a sigmoidal curve or function, was found in generally most of the charts. The effects of each individual extract on all the cell lines are shown in [Figure 1]. IC50 values of the extracts and the positive control are synopsized in [Table 1].
Figure 1: Dose-response curves showing the cytotoxic activities of the crude leaf, root, stem, and pod extracts on the cell viability of H69PR, MCF7, THP1, HCT116, HT29, and HDFn.

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Table 1: Cytotoxic activities (IC50) of the crude leaf, root, stem, and pod extracts and Zeocin against H69PR, MCF-7, THP-1, HCT-116, HT-29, and HDFn

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The effects of each individual extract on all the cell lines are shown in [Figure 1]. Data are shown as mean ± SEM. GraphPad Prism 7.01 was used to perform extra sum-of-squares F-test to evaluate the significance of the best-fit-parameter (half maximal inhibitory concentration) among different treatments, and to determine the differences among the dose-response curve fits. The F-test for the leaf extract was F (5, 132) = 375.1, P< 0.0001 and the curve fit was F (5, 42) = 0.7269, P = 0.564. The root extract showed that the sum-of-squares was F (5,132) = 165.3, P< 0.0001 and that the dose-response was F (5, 42) = 0.9284, P = 0.4723. The F-test for the stem extract dictated that F (5, 132) = 124.4, P< 0.0001 and the curve fit to be F (5, 42) = 0.9286, P = 0.4722. Analyses of the pod extract exhibited F (5, 132) = 217.6, P< 0.0001 and the curve fit was F (5, 42) = 0.4941, P = 0.7788. The positive control Zeocin gave F (5, 132) = 1.08, P = 0.3742 and for the dose-response F (5, 42) = 0.2554, P = 0.9347.

Crude extract of the leaves gave the highest efficacy toward H69PR with a half maximal inhibitory concentration of 3.50 mg/mL, which was followed by the stem and root extracts which gave IC50 values of 4.32 and 6.35 mg/mL, respectively. The pod extract was moderately cytotoxic and was only able to decrease cell viability by 50% at 28.24 mg/mL. No significant differences, as found in Tukey's post hoc multiple comparisons, was found in all the paired treatments (P > 0.05).

Within the MCF-7 subset, the leaf extract demonstrated considerable activity as exhibited by an IC50 value of 4.02 mg/mL, followed by the activity of pod, stem, and root extracts with IC50 values of 4.91, 14.80, and 15.37 mg/mL, respectively. No significant differences, as found in Tukey's post hoc multiple comparisons, was found between all the paired treatments (P > 0.05).

Trials using THP-1 showed that the root extract gave the lowest IC50 value of 3.42 mg/mL while the leaf (IC50 =3.53 mg/mL) and stem (IC50 =3.94 mg/mL) extracts gave comparable attenuated results. The half maximal inhibitory concentration of the pod extract was 30.13 mg/mL, the highest concentration for all the trials using THP-1. Paired treatments displayed no significant differences (P > 0.05) as found in Tukey's post hoc multiple comparisons.

The leaf extract exhibited marked antiproliferative activity on HCT-116 with an IC50 value of 3.48 mg/mL. The stem, pod, and root extracts following suit with IC50 values of 3.73, 6.07, and 7.73 mg/mL, respectively. Post hoc analysis obtained that there were no significant differences between the paired treatments (P > 0.05).

HT-29 was most vulnerable to the pod extract with an IC50 value of 4.21 mg/mL. Inhibition of the viability of HT-29 cells on the leaf, stem, and root extracts was only established at IC50 values of 8.39, 8.80, and 8.92 mg/mL, respectively. The data subsets of the crude extracts and HT-29 also exhibited similar paired treatments (P > 0.05).

All the extracts were established not to be cytotoxic to wild-type HDFn, with IC50 values of the leaf, root, and pod extracts substantially surpassing concentrations of 100 μg/mL while the stem extract exhibited a lower IC50 value of 72.22 mg/mL. Multiple comparisons between all of the plant parts showed insignificant differences whereas trials with each crude extract paired with Zeocin were found to be significantly disparate (P< 0.0001). Zeocin, a known cytotoxic agent, gave IC50 values of 4.20, 3.94, 5.00, 4.12, 4.02, and 3.50 μg/mL for H69PR, MCF-7, THP-1, HCT-116, HT-29, and HDFn, respectively, and multiple comparisons showed no significant difference among the trials (P > 0.05). Dose-response curves showing the cytotoxic activities of the crude leaf, root, stem and pod extracts and Zeocin on the cell viability of H69PR, MCF7, THP1, HCT116, HT29, and HDFn [Figure 1]. Post hoc comparison of the dose-response curve fits for most of the cell line treatments exhibited no significant differences, with the exception of the analyses of HDFn trials with the crude extracts which were statistically different (P< 0.05) [Figure 1].

Assessment of the effectiveness of crude extracts on the four immortalized cancer cell lines revealed that the integrity of HCT-116 cells and HT-29 consistently decreased in all the trials and the action of cytotoxicity was analogous to the positive control Zeocin. For the most part, H69PR and THP-1 cells also followed this trend of degeneration with the exception of the pod extract which exhibited moderate cytotoxicity with IC50 results of 28.24 and 30.13 mg/mL, respectively. Trials observed in HT-29 confirmed low half maximal inhibitory concentrations (<9 mg/mL) which were acquired for all of the trials. MCF-7 was responsive to the leaf and pod extracts, but stem and root extracts were found to be damaging to breast adenocarcinoma at a higher cytotoxicity index. With regard to the efficacy of each plant part, the leaf extract was overall the most effective toward the mutant cell lines: HCT-116, H69PR, THP-1, MCF-7, and HT-29 with IC50 values of 3.48, 3.50, 3.53, 4.02, and 8.39 mg/mL, respectively. The root extract was most effectual against THP-1 with an IC50 value of 3.42 and was found to achieve effectual cytotoxicity to the other cell lines at IC50 values ranging from 6.35 to 15.37 mg/mL. The stem extract showed the highest antiproliferative effects on HCT-116, followed by THP-1, H69PR, HT-29, and MCF-7. The pod extract had highest efficacy toward HT-29 (IC50 =4.21 mg/mL), followed by the cell lines MCF-7, and HCT-116, which resulted in IC50 values of 4.91 and 6.07 mg/mL, respectively; and was cytotoxic to H69PR and THP-1 (IC50 <30.50 mg/mL).

The stipulated active cytotoxic limits of natural products are 20 mg/mL or less for crude extracts and 4 mg/mL or less for pure compounds,[21] and compounds that have been purified which demonstrate active cytotoxicity can be deemed to have promise for drug development.[22] The findings in this work disclosed that the crude extracts from A. paniculata can be candidates for chemotherapeutic drugs or used as a corollary for medical protocols in dealing with the management of small cell lung carcinoma, human breast adenocarcinoma, human acute monocytic leukemia, human colon and colorectal cancer, and human small cell lung carcinoma.

This research displayed that the cytotoxicity of the crude extracts was reliant on the particular immortalized cancer cell line used. Tukey's multiple comparisons of the two colon carcinomas showed that HCT-116 was similar in their response to all of the extracts (P > 0.05). A key consideration in the development of drugs with specific molecular targets is the verification of the exact target mechanism of the agent in intact cells or tumors.[23] In a previously reported research, the pure compounds of A. paniculata were less effective compared to dichloromethane or methanolic extract in terms of their immunomodulatory potency, which indicated that molecules other than the reported diterpenes may have also contributed to the immunostimulation or that synergistic interactions among the different constituents were in play.[24] It was also found that A. paniculata extract stimulated both antigen-specific and nonspecific immune system in mice and that the crude extract was more active than the isolated compounds.[1] Although disparity of the cell lines such as tumor heterogeneity can exhibit variance in drug-related response due to the modification and differences in expression profiles as found in cell lines such as HCT-116 and HT-29,[25] it can be assumed that the varying constituents were able to target defined molecular targets linked to the malignant phenotype.


   Conclusion Top


The dichloromethane extracts of the four parts of A. paniculata exhibited different cytotoxic activities against H69PR, MCF-7, THP-1, HCT-116, and HT-29. The comparison of the cytotoxicity activity of the crude leaf, root, stem, and pod extracts was highest for THP-1 with an IC50 value of 3.42 μg/mL for the root extract, followed by HCT-116, H69PR, and THP-1 with an IC50 value of 3.48, 3.50, and 3.53 μg/mL, respectively, for the leaf extract and HCT-116 with an IC50 value of 3.73 μg/mL for the stem extract. The leaf extract was found to be most degenerative to HCT-116 (IC50 =3.48 μg/mL), the root extract was most cytotoxic to THP-1 (IC50 =3.42 μg/mL), the stem extract was most damaging to HCT-116 (IC50 =3.73 μg/mL), and the pod extract exhibited highest efficacy with HT-29 (IC50 =4.21 μg/mL). Post hoc multiple analyses exhibited no significant difference (P > 0.05) in the dose-response curve fits as found by Brown-Forsythe test of the cytotoxicity of the crude extracts on all the mutant and normal cells. Furthermore, the extra sum-of-squares F-test used to evaluate the significance of the best-fit parameter of the half maximal inhibitory concentration among different treatments was found to be different for each data set in all the cell lines (P< 0.0001). The leaf, root, and pod extracts were not found to be cytotoxic to normal HDFn cells with IC50 values of > 100 μg/mL while the stem extract exhibited an IC50 value of > 70 μg/mL.

Discrete mechanisms governing cancer cell disruption have been found to be dependent on the immortalized cell line used. Signaling pathways in H69PR have been associated with multidrug resistance protein (MRP1) (and related proteins) which functions as a primary active transporter of numerous endo- and xeno-biotics by Phase II conjugating enzymes, such as glutathione S-transferases (GST).[26] It is possible that in H69PR, chemotherapeutic agents such as diterpenes and phenolic compounds may have acted as stimulators of GST production which can through two self-regulating pathways alleviate oxidative stress and initiate the removal of mutagenic xenobiotics. Our results showed that H69PR was highly susceptive to the leaf, root, and stem, which we reported to either have diterpenes or skullcapflavone and moderately cytotoxic to pod extracts, which we found to primarily contain phytosterols, lupeol, and triacylglycerols. THP-1 also exhibited similar activity to that of H69PR. Deterioration of monocytic THP-1 could be through interference of the PI3K-Akt pathway which augments LPS-induced availability of the mitogen-activated protien kinase pathways (1/2-extracellular signal-regulated kinases, p38 and c-Jun N-terminal kinases) and the downstream targets AP-1 and Egr-1.[27] The inhibitory mechanism found in THP-1 growth is through the PI3K-Akt pathway which influences transient regulation of strong inflammatory mediators such as nuclear factor-kappa B and glycogen synthase kinase-β (GSK-3 β).[27]

For MCF-7, HCT-116, and HT-29, the reticular activating system (RAS)/rapidly accelerated fibrosarcoma/MEK/mitogen-activated protein kinases (MAPK) signaling cascade was reported to transmit signals from their receptors to regulate gene expression.[28] The known breast cancer signaling pathways are through inhibition of fusion gene breakpoint cluster region – Abelson, Philadelphia chromosome, RAS, and TP53.In vitro and in vivo models have associated A. paniculata with downregulation of P13 kinase accompanied by Akt activation and decreased regulation of proangiogenic compounds such as osteopontin and vascular epidermal growth factor production.[29] In colon cancer, discrete mechanisms correlated with prevention of aberrant cell proliferation are RAS, deleted in colorectal carcinoma protein, mismatch repair pathways, microsatellite instability, complex protein cyclin D and cyclin-dependent kinase complex, Bad, and p53.[30] From our results, MCF-7, HCT-116, and HT-29 cell viability significantly decreased which could indicate that the constituents present could have inhibited the MAPK pathway which would have prevented proliferation. Differences in the cytotoxicity activity observed in the cell lines used can be associated with the mutations linked to cancer targeted and the pathways that could have been circumvented.

The phytosterols, diterpenes, triterpenes, carotenoid, isoprenoid alcohol, and flavone we earlier reported from the crude extracts of A. paniculata[16],[17],[18] and were previously reported to exhibit cytotoxic activities could contribute to the cytotoxic action of these extracts against H69PR, MCF-7, THP-1, HCT-116, and HT-29 immortalized mutant cell lines.

Acknowledgment

A research grant from De La Salle University Science Foundation, through the University Research Coordination Office, is gratefully acknowledged.

Financial support and sponsorship

This study was financially supported by De La Salle University Science Foundation through the University Research Coordination Office.

Conflicts of interest

There are no conflicts of interest.

 
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