Apoptotic and antimetastatic activities of betulin isolated from Quercus incana against non-small cell lung cancer cells

Figures & data

Figure 1 Structure of compounds (1–4).

Figure 2 (A) Cytotoxic effects of betulin from Quercus incana against NSCLC and normal cells at various concentrations. ^*P<0.05 was considered as significant. **P<0.01; ***P<0.001. Morphological changes in (B) NCI-H460 cells (C) NIH-3t3 cells after 24 and 48 hours of treatment with betulin, observed under phase contrast microscope at 10× magnification. (D) Effect of betulin on colony formation of H460 cells after 48 hours treatment of betulin. Cells were supplied with fresh culture media every third day and were incubated at 37°C in humidified 5% CO₂ environment for 15 days after the treatment. (E) Immunocytochemical analysis of expression of proliferative marker Ki-67 after 48 hours of treatment with betulin, observed under fluorescent microscope at 20× magnification.

Abbreviation: NSCLC, nonsmall cell lung cancer.

Table 1 IC₅₀ values of the compounds from chloroform fraction of Quercus incana against NSCLC and normal cells

Name/compound no.	NCI-H460 (μM)	NIH-3T3 (μM)
Cirsimaritin (1)	26.23±0.053	38.84±0.037
Eupatorin (2)	37.50±0.070	75.09±0.086
Betulin (3)	11.55±0.037	50.54±0.049
β-Amyrin acetate (4)	10.62±0.045	20.06±0.073
Cisplatin (standard)	19±1.24	–

Abbreviation: NSCLC, nonsmall cell lung cancer.

Figure 3 (A) Effects of betulin on the migration of NCI-H460 cells. Using phase contrast microscope at 10× magnification, scratch areas were photographed and area of the wound was calculated using Image J. (B) Percent closure of the wound after 24 and 48 hours of the treatment. In control well, the rate of migration of the cells toward the wound was increased to 41% after 48 hours in treated groups cells migration was significantly reduced. **P<0.01 vs control. ^*P<0.05; ***P<0.001. (C) Effects of betulin on invasion of NCI-H460 cells using transwell assay. Membrane was photographed under bright field at 4× magnification using inverted microscope. (D) Effects of betulin on expression levels of MMP-2 and -9 in NCI-H460 cells after 48 hours treatment as evaluated by RT-PCR. (E) Gelatinolytic activity of MMP-2/-9 in response to the treatment with different concentrations of betulin as analyzed by zymography.

Abbreviation: MMP, matrix metalloproteinases.

Figure 4 (A) Betulin (3) induced apoptosis in NSCLC cell line in a dose-dependent manner. Flow cytometric analysis of the apoptosis induced by the indicated concentrations of the compound. An increased number of cells were observed in treated groups compared to control. (B) Graphical representation of the cells in different phases of apoptosis after 48 hours of treatment. **P<0.01 compared to the untreated control. ***P<0.001. (C) Effects of betulin (3) on the mitochondrial membrane potential (Δψm) of the cells after 48 hours. Following the treatment with different concentrations, an increase in positive green fluorescent cells was observed, indicating loss of Δψm compared to the control. (D) Histogram shows the percentages of the cells with disrupted Δψm in treated groups. ***P<0.001 vs the untreated control. (E) Effects of betulin (3) on the nuclear morphology of NCI-H460 cells. Increase in DNA condensation and change in the shape of the nucleus of the treated groups have been observed in a dose and time-dependent manner compared to the control.

Abbreviation: NSCLC, nonsmall cell lung cancer.

Figure 5 (A) Analysis of effects of the compounds on the mRNA expression of cell survival and apoptotic genes. An upregulation was observed in genes of caspases family and proapoptotic genes ie, HRK, BAX and BAK, while anti-apoptotic BCL-2L1 and p53 genes and cell survival VEGF and COX-2 genes significantly downregulated. Interestingly, initiator caspase-8 of death receptor pathway is also inhibited after the treatment with betulin. GAPDH was used as reference gene. (B) Quantitative analysis of expression of apoptotic genes compared to control. ^*P<0.05 compared to control. **P<0.01; ***P<0.001. (C) Immunocytochemical analysis of major apoptotic markers after 48 hours treatment with betulin observed under fluorescent microscope at 20× magnification. The caspase-3 and caspase-6 are highly expressed in treated cells while caspase-8 is downregulated, suggesting activation of intrinsic pathway of apoptosis. Furthermore, anti-apoptotic BCL-2L1 show marked inhibition posttreatment.

Abbreviation: Int Den, integrated density.

Figure 6 (A) The gene expression analysis of osteopontin in response to the treatment with betulin after 48 hours as observed via RT-PCR. The results show pronounced downregulation of expression of the marker in comparison to GAPDH, the reference gene. (B) Proteomic analysis of osteopontin compared with the untreated control studied through immunofluorescence assay under fluorescent microscope at 20× magnification. The expression of osteopontin in the cells after the treatment. (C) Proposed mechanism of osteopontin as central regulator of apoptosis and metastasis in H460 cells.

Abbreviation: MMP, matrix metalloproteinases.

Figure 7 Synergistic effects of betulin with standard drug cisplatin as represented by (A) Fa-CI plot and (B) classic isobologram showing data points of individual drugs vs actual data points of their combination at different inhibitory concentrations observed through MTT assay.

Abbreviations: Fa, fraction affected; CI, combination index.

Figure S1 Effects on protein expression of (A) BCL-2L1, (B) caspase-3, (C) caspase-6, (D) caspase-8, (E) Ki-67, and (F) osteopontin after 48 h treatment with betulin at different concentrations.

Notes: DAPI staining indicates nuclei of cells, whereas FITC/TxR image shows expression of the respective marker under fluorescent microscope at 20× magnification.

Table S1 Primer sequences of the genes used for RT-PCR.

Gene	Sequence	Annealing temperature	References
Caspase-3	R: CAAGCTTGTCGGCATACTGTTTCAG F: TTCAGAGGGGATCGTTGTAGAAGTC	52°C	Hanif et al,^Citation1 2014
Caspase-6	R: AATTGCACTTGGGTCTTTGC F: ATCCTCACCGGGAAACTGTG	54°C	Alharbi,^Citation2 2015
Caspase-8	R: GGATGAGGCAGACTTTCTGC F: GGCCTCCATCTATGACCTAG	58°C	This study
Caspase-9	R: GCACCACTGGGGGTAAGGTTTTCTAG F: ATGGACGAAGCGGATCGGCGGCTCC	60°C	Krisnamurti et al,^Citation3 2016
BAX	R: GGCCCCAGTTGAAGTTGC F: AAGAAGCTGAGCGAGTGTC	54°C	Hanif et al,^Citation1 2014
BAK	R: CCTGAGAGTCCAACTGCAAA F: GGTCCTGCTCAACTCTACCC	60°C	Guan et al,^Citation4 2010
BCL-2L1	R: ATGGTCAGTGTCTGGTCATT F: TTGTGGAACTCTATGGGAAC	57°C	Choi et al,^Citation5 2015
p53	R: CTCTCGGAACATCTCGAAGCG F: GCTCTGACTGTACCACCATCC	57°C	Suo et al,^Citation6 2015
HRK	R: CCCCCRAAAATTAAAAAAAAAACT F: GAGTTGAATTTAGGAAAAGGGGAA	58°C	Zaker et al,^Citation7 2016
Osteopontin	R: ACGGCTGTCCCAATCAGAAG F: AGCAGAATCTCCTAGCCCCA	58°C	This study
VEGF	R: GCTTTCGTTTTTGCCCCTTTC F: CAACATCACCATGCAGATTATGC	57°C	Wang et al,^Citation8 2015
COX-2	R: GCATCTGGCCGAGGCTTTTCTAC F: TGTGGGGCAGGAGGTCTTTGGTCT	57°C	Kim et al,^Citation9 2011
MMP-2	R: GGCATCCAGGTTATCGGGGA F: GGCCCTGTCACTCCTGAGAT	59°C	Benus et al,^Citation10 2005
MMP-9	R: GCACAGTAGTGGCCGTAGAA F: CTATGGTCCTCGCCCTGAAC	54°C	This study
GAPDH	R: GGTCTACATGGCAACTGTGA F: ACGACCACTTTGTCAAGCTC	59°C	Shi et al,^Citation11 2012

Abbreviation: MMP, matrix metalloproteinases.

HanifFPerveenKJawedHN-(2-hydroxyphenyl)acetamide (NA-2) and Temozolomide synergistically induce apoptosis in human glioblastoma cell line U87Cancer Cell Int201414113314225663820

 PubMed Web of Science ®Google Scholar

AlharbiRHOX/PBX interaction as a therapeutic target in Acute Myeloid LeukemiaPhD thesisGuildfordUniversity of Surrey2015

 Google Scholar

KrisnamurtiDGBLouisaMAnggraeniEWanandiSIDrug efflux transporters are overexpressed in short-term tamoxifen-induced MCF7 breast cancer cellsAdv Pharm Sci20162016616

 Google Scholar

GuanHXieLLeithäuserFKLF4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classic Hodgkin lymphomaBlood201011691469147820519630

 PubMed Web of Science ®Google Scholar

ChoiY-CYCYoonSByunYMicroRNA library screening identifies growth-suppressive microRNAs that regulate genes involved in cell cycle progression and apoptosisExp Cell Res2015339232033226485640

 PubMed Web of Science ®Google Scholar

SuoHSongJIA-LEJLZhouYInduction of apoptosis in HCT-116 colon cancer cells by polysaccharide of Larimichthys crocea Larimichthys crocea swim bladderOncol Lett20159297297825624917

 PubMed Web of Science ®Google Scholar

ZakerFAmirizadehNNasiriNGene expression and methylation pattern in HRK apoptotic gene in myelodysplastic syndromeInt J Mol Cell Med201652909927478805

 PubMedGoogle Scholar

WangSTuJZhouCThe effect of Lfcin-B on non-small cell lung cancer H460 cells is mediated by inhibiting VEGF expression and inducing apoptosisArch Pharmcal Res2015382261271

 PubMed Web of Science ®Google Scholar

KimBMMaengKLeeK-HKHHongSHCombined treatment with the Cox-2 inhibitor niflumic acid and PPARγ ligand ciglitazone induces ER stress/caspase-8-mediated apoptosis in human lung cancer cellsCancer Lett2011300213414421067863

 PubMed Web of Science ®Google Scholar

BenusGFWierengaATde GorterDJInhibition of the transforming growth factor beta (TGFbeta) pathway by interleukin-1beta is mediated through TGFbeta-activated kinase 1 phosphorylation of SMAD3Mol Biol Cell20051683501351015917296

 PubMed Web of Science ®Google Scholar

ShiYFuXHuaYThe side population in human lung cancer cell line NCI-H460 is enriched in stem-like cancer cellsPLoS One201273e3335822428030

 PubMed Web of Science ®Google Scholar