Research ArticleExperimental Studies

The Ethanol Crude Extraction of Cyperus Rotundus Regulates Apoptosis-associated Gene Expression in HeLa Human Cervical Carcinoma Cells In Vitro

CHIA-HSIN LIN, SHU-FEN PENG, FU-SHIN CHUEH, ZHENG-YU CHENG, CHAO-LIN KUO and JING-GUNG CHUNG
Anticancer Research July 2019, 39 (7) 3697-3709; DOI: https://doi.org/10.21873/anticanres.13518

Abstract

Background/Aim: Cervical cancer is considered poorly chemo-sensitive in women and its treatment remains unsatisfactory. Cyperus rotundus is used in Chinese medicine as a therapeutic agent for women's disease. The effects and molecular mechanisms of the ethanol extraction of C. rotundus (CRE) on cervical cancer remain unclear. We aimed to explore the mechanisms and genetic influence of CRE on cervical cancer. Materials and Methods: HeLa, human cervical cancer cells were treated with various doses of CRE and changes in cell morphology and cell viability were assessed using microscopy and flow cytometry. Finally, we performed a microarray analysis to scan related genes. Results: The treatment of CRE on HeLa cells caused morphological changes and induced chromatin condensation. DNA microarray analysis showed that CRE led to up-regulation of 449 genes and down-regulation of 484 genes, which were classified in several interaction pathways. Conclusion: CRE changed HeLa cell morphology and induced gene expression which associated with apoptosis and cell-cycle arrest. These results provide important information at the transcription level for targeting treatments of human cervical cancer.

Cyperus rotundus L. (Cyperaceae) has been extensively used in traditional medicine in Asia, Africa, and Europe. It is also known as nutgrass and is cultivated widely in tropical, subtropical and temperate regions. It is prescribed for gynecological disorders, including dysmenorrhea and irregular menstruation, while its rhizomes (rootstalks) have been used as sedatives and analgesics (1, 2). Recently, many studies have demonstrated that its rhizomes have a vast range of biological and pharmacological functions, including anti-oxidant, anti-inflammatory, anti-diabetic, anti-allergic, anti-nociceptive and anti-cancer effects (2-4). The active chemical constituents of C. rotundus are saponins, alkaloids, flavonoids (anthocyanidins, catechins, flavans, flavones, flavanonols, and isoflavane), tannins, starch, glycosides, terpenoids, sesquiterpenes, sitosterol, cyperol, ascorbic acid, polyphenols, and essential oils (α-longipinane, β-selinene, cyperene, and caryophyl leneoxide) (2, 5, 6). Recently, the C. rotundus rhizome extract exhibited a protective effect against attenuated peroxynitrite (ONOO-)-induced neurotoxicity (6) and also prevented DNA damage and cytotoxicity through its antioxidant activity in human neuroblastoma SH-SY5Y cells (7).

Moreover, the anticancer properties of C. rotundus have been a focus of research recently. The essential oil isolated from C. rotundus can suppress cell proliferation and induce apoptotic DNA fragmentation in murine lymphoblastic leukemia L1210 cells and human chronic myelogenous leukemia K562 cells (4, 8). The apoptotic activity of the different fractions of C. rotundus extract has been examined in the treatment of MDA-MB-231 breast cancer. The ethanolic/methanolic extracts display an anti-proliferative activity and induce apoptosis through increased expression of the death receptor, such as TNF-α, IFN-γ, and MAPK, while inhibiting the expression of the anti-apoptotic factor survivin. On the contrary, the water extract displayed no such properties (2, 9).

Cervical cancer is a major cause of morbidity in women worldwide, with a poor chemo-sensitivity to therapy (10). The role of chemotherapy in the treatment of cervical cancer has mainly been confined to persistent or recurrent cases following failure of surgery and/or radiotherapy. Cisplatin represents the cornerstone of chemotherapy for cervical cancer, however, it has a limited efficacy due to its side-effects and the development of resistance. The induction of apoptosis has been shown to be an efficent strategy for identifying potential therapeutic agents for cancer therapy (11).

The effects and related molecular mechanism of C. rotundus extract properties on cervical cancer are not really understood. Our aim, herein, was to evaluate the anti-cancer effects of Cyperus rotundus in vitro, including its cytotoxic effect and how it affects gene expression in HeLa human cervical cancer cells.

Materials and Methods

Chemicals and reagents. Cyperus rotundus L. was planted in Taiwan (Figure 1A) and its rhizomes (Figure 1B) were purchased from Lian He Pharmacy (Taichung, Taiwan, R.O.C.). Ethanol (95%) was obtained from Echo Chemical Co. LTD (Taichung, Taiwan). DMEM medium, fetal bovine serum (FBS), L-glutamine, and antibiotics (penicillin G and streptomycin) were purchased from Gibco BRL (Grand Island, NY, USA), DAPI from Molecular Probes (Eugene, OR, USA), and trypsin and propidium iodide (PI) from Sigma Chemical Co. (St. Louis, MO, USA). The crude extraction of Cyperus rotundus L. (CRE) was conducted using 95% Ethanol.

Cell culture. HeLa cells, a well-known human cervical cancer cell line, was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan, R.O.C.). HeLa cells were maintained in DMEM medium supplemented with FBS (10%), L-glutamine (1%), and penicillin G/streptomycin (100 Units/ml/100 μg/ml) and were maintained in a 37°C incubator under 5% CO2 and 95% air (12).

Observation of cell morphology and measurement of cell viability. HeLa cells (2×105 cells/well) were placed in 12-well plates overnight and were then treated with: i) 100, ii) 200, iii) 300, iv) 400, and v) 500 μg/ml of CRE for 48 hours. Following treatment, cell morphology was observed and photographed using a phase contrast microscope (Carl Zeiss, Axiovert 25, Oberkochen, Germany) at 200× magnification and cells were detached by trypsin, subsequently wash by PBS (phosphate buffered saline), and collected in FACS tube. This was followed by PI staining (5 μg/ml) for cell viability using flow cytometry (Becton-Dickinson, San Jose, CA, USA) as described previously (13) .

DAPI nuclear staining. The effects of apoptosis, chromatin condensation and the presence of apoptotic bodies were investigated using DAPI staining. Following treatment with the aforementioned concentrations of CRE, HeLa cells were immersed in 4% paraformaldehyde for 15 min, stained with DAPI (1 μg/ml) for 30 min at room temperature and, then the morphology of nucleus was visualized and photographed using a fluorescence microscope (Carl Zeiss, Axiovert 25, Oberkochen, Germany) at 100× magnification. Cells with condensed, fragmented and degraded nuclei were recognized as apoptotic cells (14). When cells undergo apoptosis, there is enhanced fluorescent signals in the nucleus. Ten nuclei of cells were randomly selected to quantify the fluorescence intensity of DAPI signals by using the NIH Image J software, version 1.47 (National Institutes of Health, Bethesda, MA, USA).

Generation of cDNA microarray, hybridization, and scanning for gene expression in HeLa cells following exposure to CRE. HeLa cells (6×105 cells/dish) were sub-cultured in 10-cm dish for 24 h. Then cells were treated with CRE (300 μg/ml) or not (control) for 48 h. Total RNA was extracted and purified using the Qiagen RNeasy Mini Kit (Qiagen, Inc, Valencia, CA, USA), according to manufacturer's instructions. Subsequently, cDNA was reverse transcribed from extracted RNA and then fluorescently-labeled for probe preparation. Fluorescence-labeled cDNAs were probed to their complementary strands on the chip (Affymetrix GeneChip Human Gene 1.0 ST array, Affymetrix, Santa Clara, CA, USA) and the hybridization signals were examined and quantified by Asia BioInnovations Corporation (Taipei, Taiwan, ROC). Finally, the difference of data was analyzed using the Expression Console software (Affymetrix) with default RNA parameters. Genes up- or down-regulated by at least 2-fold in CRE-treated cells compared to controls were recorded. Data are representative from three separate assays (15).

Gene ontology analysis. For detecting significantly over-represented GO (Gene Ontology) biological processes we used DAVID (Database for Annotation, Visualization and Integrated Discovery), that provides an overall set of functional annotation tools for researchers to know biological meaning behind large list of genes. Enrichment was determined at a calculated Benjamini value of <0.05. Statistical significance of overexpressed individual genes was determined using a standard Student's t-test.

Statistical analysis. Experimental values are expressed as mean±standard deviation (SD) from three independent experiments, each conducted in triplicates. Statistical significance (*p<0.05, **p<0.01, and ***p<0.001) was assessed using one-way ANOVA and Tukey multiple comparison test for comparing CRE-treated groups to the control (16).

Results

CRE induced cell morphological changes and decreased viable HeLa cells in vitro. At first, we assessed the cell viability of HeLa cells following exposure to CRE. As shown in Figure 2A, CRE treatment at high concentrations (200-500 μg/ml) significantly decreased cell viability and caused morphological changes in HeLa cells in a concentration-dependent manner (Figure 2B). CRE-treated cells showed shrunken, deformed, and small vacuoles appear inside cells. The half-maximal inhibitory concentration (IC50) for the 48-h treatment of CRE in HeLa cells was 300 μg/ml. Therefore, CRE at 300 μg/ml was selected for the following steps of our study.

Figure 1.
Figure 1.

The wild plant (A) and the dried rhizome (B) of Cyperus rotundus.

CRE induced chromatin condensations of HeLa cells by DAPI staining. For further verifying whether CRE decreased the total number of viable HeLa cells through apoptosis, cells were incubated with different concentrations of CRE for 48 hours, and were stained by DAPI to assess the formation of chromatin condensation. As shown in Figure 3A and B, the higher fluorescence intensity (DAPI staining) was the result of chromatin condensation (15) at much higher levels in CRE-treated HeLa cells compared to control. Similar to the effect of CRE in cell viability, the results from this experiment also indicated that CRE-induced chromatin condensation occurred at a concentration-dependent manner.

CRE induces up- and down-regulation of gene expression in HeLa cells. The results from the cDNA microarray analysis, regarding apoptosis, are shown in Tables I and II. Quantification and data filtration revealed 33297 native features in our dataset; however, after collapsing features into gene symbols, only 20,693 features remained. The genes included in the CRE-treated group were 10,616 with a correlation area 53.5% while the control group was 10,077 with a correlation area 46.5%. That indicates the gene expression in CRE-treated and control groups were different. Furthermore, the differences of gene expression between these two groups were compared in the HeLa cell. Table I shows 11 genes exhibiting >10-fold change and 8 genes with a >5-fold change. This list includes: i) DDIT3 and GADD45A that are genes associated with DNA damage (17), ii) CDKN1A and CDKN2B that are genes associated with the cell cycle (18, 19), iii) NCF2, ATP6V0D2, HMOX1, ATF3, and CGRRF1 genes, related to cell survival (20,21), and iv) TNFRSF21, TRAF1, IL6, and ATG13 genes, related to cell apoptosis (22-24). Table II shows genes that were downregulated. This includes the DDIAS gene, linked to DNA damage (25), while some genes, such as CDK1, CCNF, CDCA2, CCNA2, CDCA3, GTSE1, CCNE2, CDC20, and CDK2, which are associated with cell cycle (26, 27).

Table III indicates the gene ontology categories of biological processes, cellular component, and molecular function. There are 125 genes related to cell cycle and 184 genes related to the function of an intracellular non-membrane-bound organelle. Molecular function describes activities, such as catalytic reaction or binding that occurs at a molecular level. The nucleotide or nucleoside binding had the deepest impact on the gene expression of CRE-treated cells. According to the results of the BIOCARTA pathway analysis shown in Table IV, the differentially expressed genes in CRE-treated HeLa cells were related to cell cycle and cancer pathways.

The cDNA microarray analysis of CRE-treated or untreated cells revealed the results of top, second and third scores, as shown in Figures 4, 5 and 6, respectively. The analysis results are mapped on the processes presenting possible signal effects. Genes marked red are upregulated while blue-marked genes are downregulated. Circles of different intensities display different enhancement or suppression of genes in CRE-treated HeLa cells compared to control cells. Especially, some genes show up- and down-regulation to associate with apoptosis of cancer cells. NFATC2 controls melanoma dedifferentiation by inducing expression in carcinoma cells of membrane-bound tumor necrosis factor-α (mTNF-α), while melanoma-expressed TNF-α regulates a c-myc-Brn2 axis.(28). JOSD1 depletion can lead to severe apoptosis in gynaecological cancer cells both in vivo and in vitro (29). The PTGS2 gene, which encodes cyclooxygenase 2 (COX-2), is deregulated in endometriotic lesions and plays an important role in the acquisition of oocyte competence (30). IRAK is responsible for the regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation (31). Ectopic expression of MDA-5 has been shown to induce carcinoma cell death, and then intentionally targeting the evolutionarily keep MDA-5-IPS-1 antiviral pathway in tumors can cause parallel tumoricidal effect that creates a bridge between innate and adaptive immune responses for the therapeutic treatment of cancer (32). These three maps show proteins and genes that are part of pro-apoptotic pathways in cancer cells.

Figure 2.
Figure 2.

CRE induced cell morphological changes and decreased the total number of viable HeLa cells. Morphology changes of HeLa cells rounding up and condensing (red arrows) were observed and photographed under a phase contrast microscope at 200× magnification (A). All cells were stained by propidium iodide (PI) and the percentage of viable cells was calculated (B) following treatment with different concentrations of CRE (100, 200, 300, 400, and 500 μg/ml) for 48 hours.

Discussion

C. rotundus is used as a gynecological medicine in Chinese medicine. Many ancient books, such as Jingui Yaolue, Yizong Jinjian, Shanghan Lun, etc., have recorded a therapeutic formula containing C. rotundus (33). We attempted to provide some scientific explanations with regards to this traditional treatment. In this study, we evaluated the effects of CRE on apoptotic cell death and associated gene expression of HeLa human cervical cancer cells. CRE induced cell morphological changes and reduced the total number of viable cells in a dose-dependent manner. Following treatment with various doses of CRE, HeLa cells were characterized by chromatin condensation measured by DAPI staining. Chromatin condensation has been recognized to be a marker of cell death (34). To investigate whether CRE induced cytotoxic effects and cell death by regulating the expression of apoptosis-associated genes in HeLa cells, we used cDNA microarray assay.

Figure 3.
Figure 3.

CRE-induced chromatin condensation in HeLa cells. The HeLa human cervical carcinoma cells were incubated with various concentrations of CRE (100, 200, 300, 400, and 500 μg/ml) for 48 hours and chromatin condensation (apoptosis) was determined by DAPI staining (A, yellow arrows). The relative fluorescence intensity of CRE-treated cells was calculated (B). *p<0.05 shows the significant difference between CRE-treated groups and the control, as analyzed by a Student t-test.

CRE treatment promoted the expression of certain genes associated with DNA damage and cell growth arrest, such as DDIT3 and GADD45A (35, 36). Also affected by CRE, the NCF2 gene, which encodes for the neutrophil cytosolic factor 2, is the 67-kDa cytosolic subunit of the multi-protein NADPH oxidase complex and a novel p53-targeted gene (21). Reactive oxygen species (ROS) are a by-product of normal oxygen metabolism and play a large role in cell signaling, while maintaining body contingency. However, the amount of ROS can increase dramatically under the influence of time and stress from the external environment, for example, UV or heat exposure, and may have an important role in regulating signal transduction pathways (37). The cause of change in NCF2 expression may be due to significant damage caused to a cellular structure. p53 and its family members could act as upstream regulators of ROS by transcriptionally modulating genes related to cellular redox state and leading to cell death (38, 39). The expression of the NCF2 gene represents the highest fold change following CRE treatment of HeLa cells. Additional ROS-related genes, such as SOD2 and TP53INP1, were also up-regulated in this study.

View this table:
Table I.

Up-regulated genes expressions following CRE treatment of HeLa cells.

View this table:
Table II.

Down-regulation of gene expressions in CRE-treated HeLa cells.

Importantly, CRE treatment induced cell-cycle arrest in HeLa cells. CRE-treated HeLa cell increased p21 and p15 expression by a 10.52- and 3.21-fold, respectively. These two cyclin-dependent kinase inhibitors (CDKIs), p21 and p15, are proteins that bind to and inhibit the activity of CDKs and resulted in suppressed cell growth (40, 41).

View this table:
Table III.

Gene ontology categories of biological processes, cellular component and molecular function.

Interestingly, two microRNA molecules, MIR22HG and MIR222, were up-regulated by 4.88- and 2.39-fold, respectively. Non-coding RNAs (lncRNAs and microRNAs) are non-protein-coding transcripts involved in various biological functions (42). MIR22HG, the host gene for miR-22, has been shown to be upregulated in response to chemical stresses or hypoxia (43-45). The other microRNA, miR-222, plays multiple roles in promoting cell proliferation, invasion, migration, and decreases cell apoptosis, while enhancing the sorafenib resistance of HCC cells by activating the PI3K/AKT signaling pathway (46). In addition, the literature has reported that miR-221/miR-222 expression can also regulate post-transcriptionally the expression of p27Kip1 and affect the proliferation of cancer cells (47, 48). Despite this data, the molecular mechanism of miR-222 concerning CRE treatment of HeLa cells is waiting for further clarification in the future.

View this table:
Table IV.

BIOCARTA pathway analysis of the differentially expressed genes in CRE-treated HeLa cells.

Treatment with CRE also inhibited ertain gene expression profiles, such as cell cycle-associated genes CDK1 (cyclin-dependent kinase 1), WEE1 (WEE1 G2 checkpoint kinase), and CDC25A (cell division cycle associated 5). There are literature reports showing poor prognosis for gastric cancer patients with overexpressed carboxypeptidase A4 (CPA4), a zinc-containing exopeptidase (49). In this study, elevated CPA4 expression was detected in more than 50% of primary gastric cancer cases but it was weak or absent from the normal mucosa. Clinical relevance analysis has shown that CPA4 is significantly associated with tumor growth, stage, lymph node metastasis, invasion and distal metastasis. In another study, CPA4 was associated with prostate cancer aggressiveness, histone hyperacetylation pathway, and possibly the modulation of growth-affected peptides' function and the regulation of prostate epithelial cells (50). Our results showed that CRE-treated HeLa cells decreased CPA4 gene expression by 7.61-fold, which we believe provide the first report concerning the expression of CPA4 in HeLa cells.

Gene expression changes in CRE-treated HeLa cells were classified into: i) biological processes, ii) cellular components, and iii) molecular functions. Results showed that cell cycle and related processes, intracellular non-membrane-bounded organelles, and nucleoside-binding genes were associated with the CRE treatment in HeLa cells. Furthermore, the BIOCARTA pathway analysis indicated that CRE treatment affected the G1/S and G2/M cell-cycle check point, p53 and ATM signaling pathways, as well as the caspase cascade in HeLa cells.

Figure 4.
Figure 4.

The top scored network from CRE IC50 and control samples. Thick cyan lines indicate the fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles.

In summary, CRE decreased HeLa cell viability and induced chromatin condensation. The affected gene expression in CRE-treated HeLa cells were analyzed using cDNA microarray analysis, which provided complete information on the genes and pathways targeted by CRE in HeLa cells following 48 h of treatment. From these observations, we have illustrated the possible signal transduction pathways involved with the affected genes following CRE-treatment. Future investigations are needed to extend our new findings and obtain experimental evidence concerning the molecular mechanism of the identified targets following CRE treatment in cervical cancer.

Figure 5.
Figure 5.

The second scored network from CRE IC50 and control sample. Thick cyan lines indicate the fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles.

Figure 6.
Figure 6.

The third scored network from CRE IC50 and control sample. Thick cyan lines indicate the fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles.

Acknowledgements

Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China Medical University, Taichung, Taiwan, R.O.C.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Authors' Contributions

    CHL, CLK and JGC conceived and designed the experiments. CHL and SFP performed the experiments. CHL, ZYC and SFP analyzed the data. CLK and JGC contributed towards reagents/materials/analysis tools. CHL, SFP and JGC wrote the paper.

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors confirm that there are no conflicts of interest.

  • Received March 19, 2019.
  • Revision received May 14, 2019.
  • Accepted May 15, 2019.

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The Ethanol Crude Extraction of Cyperus Rotundus Regulates Apoptosis-associated Gene Expression in HeLa Human Cervical Carcinoma Cells In Vitro
CHIA-HSIN LIN, SHU-FEN PENG, FU-SHIN CHUEH, ZHENG-YU CHENG, CHAO-LIN KUO, JING-GUNG CHUNG
Anticancer Research Jul 2019, 39 (7) 3697-3709; DOI: 10.21873/anticanres.13518
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