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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 388-393

Longan flower proanthocyanidins induce apoptosis in HT-29 colorectal carcinoma spheroids


1 Department of Surgery, Cheng Ching Hospital, Chung-Kang Branch, Taichung, Taiwan, China
2 Department of Colorectal Surgery, China Medical University Hospital, Taichung, China
3 Department of Biotechnology and Pharmaceutical Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan
4 Department of Medical Laboratory Science and Biotechnology, Yuanpei University of Medical Technology, Hsinchu, Taiwan
5 Department of Medical Imaging and Radiological Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan
6 Department of Biomedical Engineering, Yuanpei University of Medical Technology, Hsinchu, Taiwan

Date of Web Publication29-Jun-2018

Correspondence Address:
Chih-Ping Hsu
Department of Medical Laboratory Science and Biotechnology, Yuanpei University of Medical Technology, No. 306 Yuanpei Street, Hsinchu City 30015
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.176170

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 > Abstract 

Aim of Study: Proanthocyanidin-rich longan flower extract (LFP) has been previously shown to inhibit the proliferation and anchorage-independent growth in soft agar of two colorectal carcinoma (CRC) cells in vitro. In this report, we further examined the effects of LFP in a CRC spheroid model.
Materials and Methods: A liquid-overlay assay employing HT-29 spheroids was used to evaluate the effects of LFP on cancer cell tumorigenesis, viability, and apoptosis. Associated effects on signaling path ways (epidermal growth factor receptor [EGFR], Akt) and apoptotic regulators were measured using Western blot.
Results: Treatment with LFP up to 200 μg/ml inhibited tumor growth in a dose-dependent manner and induced prominent apoptosis as measured by annexin V staining. Cells treated with LFP showed decreased EGFR and Akt phosphorylation with decreased expression of B-cell lymphoma 2.
Conclusion: The ability of LFP to induce apoptosis in CRC spheroids warrants further investigation of its composition and identification of tumor-active components.

Keywords: Colorectal carcinoma, HT-29, longan flower proanthocyanidins, spheroid


How to cite this article:
Chung YC, Chiang HC, Chang H, Lin CC, Lo LT, Wang AY, Chou KF, Hsu CP. Longan flower proanthocyanidins induce apoptosis in HT-29 colorectal carcinoma spheroids. J Can Res Ther 2018;14, Suppl S2:388-93

How to cite this URL:
Chung YC, Chiang HC, Chang H, Lin CC, Lo LT, Wang AY, Chou KF, Hsu CP. Longan flower proanthocyanidins induce apoptosis in HT-29 colorectal carcinoma spheroids. J Can Res Ther [serial online] 2018 [cited 2019 Oct 23];14:388-93. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/388/176170


 > Introduction Top


Some cultured cancerous cells could form cell cluster named spheroids when the cells were detached from the culture dish. The cells in spheroids would survive and grow possibly because of resistance to apoptosis or anoikis, which is the mechanism that malignant tumor avoid undergoing apoptosis when they are detached from the extracellular matrix and associated with tumor metastasis.[1],[2],[3] Tumor cell spheroids is a possible model to test in vitro anti-tumor activity of some anticancer drugs or nature products.

Colorectal carcinoma (CRC) became the most prevalent cancer over hepatocellular carcinoma in Taiwan from 2007.[4] CRC initiation and progression from colon mucosa stem cells often occur through sequential accumulation of gene mutations, chromosomal instability, and epigenetic alterations that lead the cells to transform to become invasive and metastatic malignant cells.[5] Phenolic compounds from various plant organs, such as flowers, seeds, stems, roots, and leaves, have been shown to reduce the risk of many diseases, including CRC.[6],[7],[8],[9],[10],[11]

Longan (Dimocarpus Longan Lour.), a well-known summer fruit tree in Asian subtropical areas including Thailand, Taiwan, and China, possesses commercial roles both as fruit and medicine.[12] In traditional Chinese herbal medicine, the longan reproduction parts, such as flowers, fruit, and seeds, are used for different medical applications. The fruit acts as a digestive aid to treat fever, parasitic diseases, and intoxication. The seeds can overcome sweating and as a styptic,[13] and the flowers can be applied as a drug for treating urinary tract inflammation and micturition dysfunction.[14] Recently, high levels of polyphenolics including flavonoids and proanthocyanidins have been obtained from longan flowers by a hot water reflux method and have been demonstrated to exhibit antioxidant activity, free radical scavenging activity, and anti-inflammatory properties.[15] Our previous report confirmed that longan flower proanthocyanidins (LFP) inhibited colon cancer cell lines, CRC SW480 and Colo 320DM, by inducing S phase arrest in the cell cycle, concomitantly suppressing CRC cell growth from single cells to colonies in soft agar, implying a role of LFP in CRC treatment.[16] However, it is still not fully understood whether LFP can inhibit tumor growth and restore anoikis in CRC cells.

In this study, we cultured another CRC cell line, HT-29, which is a Grade II CRC cells with a genetic wild type of oncogene k-ras and mutation of tumor suppressor p53, and formed spheroids on a liquid-overlay culture to examine the ability of LFP to overcome anoikis resistance in CRC cells. The results showed that LFP treatment decreased the volume of the spheroids and induced anoikis in HT-29 cells, implying a potential application of LFP in prevention and treatment of CRC metastasis.


 > Materials and Methods Top


Materials

All of the materials used for cell cultures, such as Roswell Park Memorial Institute (RPMI) media 1640, fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA) and antibiotics, were obtained from Gibco Ltd., (Paisley, UK). Agarose was obtained from Amresco (Solon, OH, USA). Acrylamide, proteinase inhibitor cocktail, sodium orthovanadate, sodium fluoride, sodium pyrophosphate, Triton X-100, ammonia persulfate, Tween 20, N, N, N', N'-tetramethylethylenediamine were purchased from Sigma (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce (Rockford, IL, USA). Polyvinylidene fluoride (PVDF) membrane (immobilon-P) was obtained from Millipore (Bedford, MA, USA). Mouse monoclonal anti-caspase 3 antibody, rabbit polyclonal antibodies to epidermal growth factor receptor (EGFR), Akt, phosphor-Akt (phosphor Ser473) and B-cell lymphoma 2 (Bcl-2), and rabbit monoclonal antibodies to Bcl-2 associated X protein (Bax) and phosphor-EGFR (phospho Tyr1092), goat polyclonal anti-poly (ADP-ribose) polymerase (PARP) antibody and goat anti-rabbit were purchased from GeneTex (Hsinchu, Taiwan). Annexin V conjugated with fluorescein isothiocyanate (FITC) was obtained from Gene Research (Taipei, Taiwan). Longan flowers were purchased from a certified longan farm (Tainan, Taiwan). LFP was obtained using the hot-water reflux method, and the phenolic species were determined by using the colorimetric methods as described previously,[15],[16] and approximately 6 g of LFP powder were extracted from 50 g of dried longan flowers in this study.

Cell line

Human CRC cell line HT-29 was obtained from the Bioresource Collection and Research Center, Taiwan. HT-29 was derived from Grade II colon adenocarcinoma, cultured in 90% RPMI medium 1640 with 10% heat-inactivated FBS. RPMI media were supplemented with 25 U/ml penicillin and 25 μg/ml streptomycin. The cells were incubated at 37°C in a 5% CO2 humidity atmosphere. All experiments were carried out on a cell line-passaged 5–20 times.

Liquid-overlay culture

For spheroid growth, HT-29 cell suspensions were dispensed onto 0.8% agarose-coated dishes. The medium was half replaced every 3 days. Observation of the morphology of formed spheroids was carried out using an inverted microscope (Nikon, Eclipse TS100, Japan).

Spheroid growth curve

Spheroid images, at least, 10 different spheroids of each experimental group were captured using an inverted microscope (as previously described) equipped with a CCD camera (Imaging Source, DFK 41AU02, Germany) and measured using image analysis software on a micrometer scale. The scale of the images is 1.8 pixel/μm. Two radii of each spheroid were used to calculate the volume (μm 3) according to V = 4/3 π (ab) 3/2, where a and b were the minimum and maximum radii.[17]

Cell survival rate

HT-29 spheroids obtained from the liquid-overlay culture on the 12th or 21st day as described above were treated with different concentrations of dimethyl sulfoxide-dissolved LFP (0, 20, 40, 80, 100 or 200 μg/ml). The volumes of the spheroids were estimated, and the growth curves were calculated as described above. To evaluate the cell survival rate, cells in spheroids after 48 h of LFP treatment were trypsinized, dispersed into a single-cell suspension, and stained with trypan blue. The number of cells was counted in duplicate using a hemocytometer. The data used were the average of three independent experiments.

Apoptosis analysis

Apoptosis analysis was performed using annexin V to label cell surface phosphatidylserine (PS) of apoptotic cells.[18] Briefly, spheroid cells were trypsinized and dispersed into single-cell suspensions. The cells were washed twice with phosphate-buffered saline (PBS), followed by suspension in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2). Cells were stained with annexin V conjugated with FITC at a final concentration of 2 μg/ml at room temperature in the dark for 30 min. Flow cytometry was used to measure the fluorescence intensity, with the FL-1H channel detecting FITC. Untreated cells served as a negative control.

Immunoblotting

The protein profile change in LFP-treated CRC cells was assessed by immunoblotting as described previously.[18] Briefly, ice-cold PBS-washed cells were lysed in homogenization buffer (10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100 proteinase inhibitor cocktail) on ice for 30 min and then centrifuged at 15,000 g for 30 min at 4°C to remove insoluble materials. The protein concentration in the cell lysate was determined using a BCA protein assay kit. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resolved proteins were electrotransferred to PVDF membranes using semi-dry blot apparatus (Trans-Blot SD Semi Dry Transfer Cell, Bio-Rad). The PVDF membranes, after blocking the residue free protein binding sides with 5% fat-free milk in Tris-buffered saline supplemented with Tween 20 (TBST; 10 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Tween 20), were incubated with primary antibody in 3% fat-free milk in TBST at 4°C for 18 h. The membrane was incubated with secondary antibody conjugated with horseradish peroxidase, and the images were developed using enhanced chemiluminescence. The chemiluminescence images were photographed, and the intensities of the protein bands were quantitated (Bio-Rad GelDoc XR).

Statistical analysis

Data are expressed as mean values ± standard error of means or standard deviations unless stated otherwise. Differences between groups were calculated using Student's unpaired t-test. P <0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).


 > Results Top


HT-29 spheroid growth

The anchorage-independent growth of HT-29 cells formed clustered tight spheroids in a liquid-overlay culture. The form of the spheroids could be observed under a microscope after culturing for 3 days. The spheroids grew continuously and reached a maximum volume at 29 days, then shattered into cell clumps after that point [Figure 1]a. The volume of the HT-29 spheroids was <2.0 × 105 μm 3 at day 3, and the spheroids grew exponentially from day 3 to day 16, on which the volume was approximately 2.0 × 106 μm 3. The growth of the spheroids slowed after day 17, and they approached the maximum size at day 29, with a volume >3.5 × 106 μm 3 [Figure 1]b.
Figure 1: Tumor spheroid growth: HT-29 cells in a liquid-overlay culture. (a) Representative images of HT-29 spheroids grown on agar-coated dishes at the indicated time points. Scale bar: 100 μm. (b) HT-29 spheroid growth curve. Agar-coated dishes were used to generate HT-29 spheroids. Images were obtained using an inverted microscope. The analysis was carried out using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and growth curves were obtained. Volume values from at least 10 spheroids are averaged and represented as means ± standard deviation

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Inhibition of spheroid growth by longan flower proanthocyanidins

The inhibitory effect of LFP on HT-29 spheroid growth in terms of volume was first investigated. We treated 12th-day spheroids of HT-29 cells with various concentrations of LFP for 24 and 48 h. As shown in [Figure 2]a, the size of the spheroids decreased gradually with an increasing LFP concentration as compared with untreated spheroids [Figure 2]a. The growth curve in terms of volume of the LFP-treated spheroids is shown in [Figure 2]b, and decreased significantly in the groups of all LFP concentrations after 48 h [Figure 2]b. An inhibitory effect of LFP on 21st-day spheroids, larger spheroids than those observed on the 12th day, was also observed; however, there was no statistical difference between the LFP-treated and control spheroids [Figure 2]c.
Figure 2: Longan flower proanthocyanidins influence on tumor spheroid growth. (a) Twelfth-day HT-29 spheroids were treated with increasing concentrations of longan flower proanthocyanidins as indicated then incubated for 24 and 48 h. Representative images after 24 h of treatment (a) and calculated average volume values from at least 10 spheroids at 24 h and 48 h (b) are shown. The growth curves of longan flower proanthocyanidins-treated 21st-day spheroids are shown in (c). The results are presented as means ± standard error of mean. *P <0.05, #P <0.01. Scale bar: 100 μm

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Inhibition of spheroid cell survival

We dispersed the cells in 12th-day spheroids treated for 48 h with LFP and investigated the cell survival rate. As shown in [Figure 3], compared with the untreated control, the survival rate decreased gradually with >50% inhibition at 40 μg/ml and <40% inhibition at 80 μg/ml (P < 0.05).
Figure 3: The survival rate of longan flower proanthocyanidins-treated HT-29 spheroid cells. 12th-day HT-29 spheroids were treated with increasing concentrations of longan flower proanthocyanidins as indicated and then incubated at 37°C for 48 h. Spheroid cells were trypsinized, dispersed and stained with trypan blue and counted under a microscope. Cell viability is expressed as a percentage of untreated cells. Data are represented as mean values ± standard deviation. *P <0.05, #P <0.01

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Longan flower proanthocyanidins-induced spheroid early apoptosis

LFP-induced spheroid early apoptosis was assessed by PS translocation to determine the apoptosis of LFP-treated HT-29 spheroid cells by staining with FITC-conjugated annexin V. The number of annexin V-positive cells increased in a dose-dependent manner [Figure 4]; P < 0.05], with more than 30% cells at 40 μg/ml and 40% at 80 μg/ml.
Figure 4: Apoptosis of longan flower proanthocyanidins-treated HT-29 spheroid cells. Twelfth-day HT-29 spheroids were treated with increasing concentrations of longan flower proanthocyanidins as indicated and then incubated at 37°C for 48 h. Spheroid cells were trypsinized, dispersed and stained with annexin V conjugated with fluorescein isothiocyanate. Ten thousand cells were analyzed by flow cytometry. Data are represented as mean values ± standard deviation. *P <0.05

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Levels of apoptosis-associated proteins

The protein or phosphorylation levels of LFP-treated spheroids were as shown in [Figure 5]a. The phosphorylation level of EGFR was gradually decreased in LFP-treated spheroids, whereas the protein level of EGFR was not altered as compared with the control. The expression and phosphorylation level of Akt were found to decrease gradually with increasing LFP concentration. The Bax level was not altered, but the Bcl-2 level was decreased in LFP-treated spheroids. The pro-caspase 3 level in LFP-treated HT-29 spheroid cells gradually decreased and cleaved caspase 3 increased. The PARP level was also decreased, and cleaved PARP increased at 40 μg/ml. The Bax: Bcl-2 ratio was gradually increased and was significantly different in spheroids under 40 μg/ml and 80 μg/ml LFP treatment [Figure 5]b.
Figure 5: Immunoblots of apoptosis-linked proteins in longan flower proanthocyanidins-treated HT-29 spheroids. Twelfth-day HT-29 spheroids were treated with increasing concentrations of longan flower proanthocyanidins as indicated and then incubated at 37°C for 48 h. Cell protein lysates from HT-29 spheroids were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and immunoblotted to show epidermal growth factor receptor, phosphorylated epidermal growth factor receptor, Akt, pAkt, B-cell lymphoma 2 associated X protein, B-cell lymphoma 2, caspase 3 and poly (ADP-ribose) polymerase, with the beta-actin level used as the loading control. Protein levels were quantified using Image Lab™ Software (Bio-Rad) according to the density of each band on the immunoblotting image, normalized to the reference band (β-actin) and presented as the fold of the untreated control (a). The ratio of B-cell lymphoma 2 associated X protein to B-cell lymphoma 2 was calculated by quantifying the protein levels according to the density of the immunoblotting image (b). Data are represented as mean values ± standard deviation. *P <0.05, #P <0.01

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 > Discussion Top


In this study, we demonstrated that LFP is capable of suppressing exponentially-growing spheroids and restoring anoikis in CRC HT-29 cells. Moreover, LFP treatment elevated the Bax: Bcl-2 ratio and activated caspase 3. These observations were a result of the suppression of EGFR phosphorylation and its downstream signaling Akt phosphorylation and expression level. In our previous report, it was shown that LFP is capable of inhibiting colony formation from single cells in two colorectal cancer cell lines, SW480, and Colo 320DM, in soft agar,[16] indicating a possible role of LFP in disturbing the anchorage-independent growth of CRC. However, the kinetics of the LFP function in single cells may differ from those in multilayer tumor cells. In this study, an overlay-liquid culture was employed to mimic a tumor-like cell cluster for analysis of the LFP effect on CRC tumors and anoikis. We found that the size and growth conditions were major factors modulating the effect of LFP. When the tumor growth rate was in the exponential phase, and the volume was lower than 1.0 × 106 μm 3, LFP efficiently inhibited the growth of spheroids at the concentrations used in this study. However, the efficiency of LFP to inhibit spheroid growth was found to be limited when the spheroids enlarged to approximately 3.0 × 106 μm 3 since the spheroids at this stage may consist predominantly of quiescent cells with reducing LFP uptake, whereas small spheroids may consist more proliferating cells, as described previous report.[19] The results suggested that the application of LFP in CRC may be effective in small CRC tumors.

The molecular mechanisms of apoptosis or anoikis resistance of CRC cells include EGFR signaling,[20] which may be caused by integrin activating pro-survival pathways (PI-3 kinase/Akt) and results in ligand-independent activation. HT-29 cells can survive in anchorage-independent conditions and contain few apoptotic cells, indicating resistance to apoptosis. In contrast, LFP-treated HT-29 cell spheroids exhibited suppressed growth and more apoptotic cells, implying that LFP could surmount the resistance to apoptosis of HT-29 cells. Furthermore, LFP-treated HT-29 spheroids exhibited suppressed Bcl-2 level and caspase 3 activation, suggesting the restoration of apoptosis of HT-29 by LFP treatment. The changes in apoptosis mechanisms may result from disturbance of EGFR phosphorylation by LFP. Our results revealed that LFP was capable of downregulating EGFR phosphorylation/activation, concomitantly suppressing both the expression and phosphorylation level of Akt, and simultaneously triggering apoptosis of HT-29 spheroids. It is noted that as previous report,[16] k-ras mutated SW480 cells could not be induced apoptosis by LFP treatment, but could suppress the clonogenic growth in soft agar from single cell. This further indicates the important role of disturbing EGFR on LFP-induced apoptosis or may be anoikis. The results suggested a potential role of LFP in targeting EGFR signaling and overcoming the apoptosis resistance of CRC cells.


 > Conclusion Top


We highlight possible roles of LFP in the restoration of apoptosis in CRC HT-29 cells. A possible mechanism is that LFP disturbs EGFR phosphorylation and suppresses Bcl-2 expression. The active ingredients of LFP responsible for targeting EGFR signaling and restoring apoptosis in colorectal cancer cells should be investigated in future studies. LFP possesses the ability to inhibit EGFR signaling and restore apoptosis in CRC cells. Our results suggested that LFP has the potential for medical application for the prevention and treatment of CRC cells in the future.

Financial support and sponsorship

This work was supported by grants from Cheng-Ching General Hospital (CH10200154A) and the Ministry of Science and Technology (MOST 103-2632-E-264-001), Taiwan.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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