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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.45 No.4 pp.152-161

Anticancer effects of D-pinitol in human oral squamous carcinoma cells

Hyun-Chul Shin1, Tea-Hyun Bang1, Hae-Mi Kang1, Bong-Soo Park1,2, In-Ryoung Kim1,2*
1Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
2BK21 FOUR Project, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
*Correspondence to:In-Ryoung Kim, E-mail:
October 14, 2020 December 10, 2020 December 10, 2020


D-pinitol is an analog of 3-methoxy-D-chiro-inositol found in beans and plants. D-pinitol has anti-inflammatory, antidiabetic, and anticancer effects. Additionally, D-pinitol induces apoptosis and inhibits metastasis in breast and prostate cancers. However, to date, no study has investigated the anticancer effects of D-pinitol in oral cancer. Therefore, in this study, whether the anticancer effects of D-pinitol induce apoptosis, inhibit the epithelialto- mesenchymal transition (EMT), and arrest cell cycle was investigated in squamous epithelial cells. D-pinitol decreased the survival and cell proliferation rates of CAL-27 and Ca9-22 oral squamous carcinoma cells in a concentration- and time-dependent manner. Evidence of apoptosis, including nuclear condensation, poly (ADP-ribose) polymerase, and caspase-3 fragmentation, was also observed. D-pinitol inhibited the migration and invasion of both cell lines. In terms of EMT-related proteins, E-cadherin was increased, whereas N-cadherin, Snail, and Slug were decreased. D-pinitol also decreased the expression of cyclin D1, a protein involved in the cell cycle, but increased the expression of p21, a cyclin-dependent kinase inhibitor. Hence, D-pinitol induces apoptosis and cell cycle arrest in CAL-27 and Ca9-22 cells, demonstrating an anticancer effect by decreasing the EMT.


    National Research Foundation of Korea(NRF)
    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


    The term “head and neck cancer” is used to describe various types of cancers, including cancer of the oral cavity, paranasal sinuses, pharynx, and larynx. Additionally, the term “oral cancer” broadly encompasses tumors arising in the lips, hard palate, alveolar ridges, tongue, sublingual region, and buccal mucosa [1,2]. More than 90% of all oral cancers are squamous cell carcinomas that arise from the squamous epithelial cells that make up the mucous membranes of the mouth [3,4]. In addition, sarcoma of the facial soft tissue and malignant melanoma, and more rarely lymphoma, can appear in cases of salivary gland carcinoma [4]. The overall five-year survival rate, including all the stages of oral cancer, is approximately 50%, and both local and distant metastases are found in more than 70% of patients at the time of diagnosis [5]. The incidence of oral cancer has been increasing worldwide for many years, and most cases are treated by means of oral surgery because the facial area is an exposed and thus easily accessible part of the body. The use of cosmetic surgery for reconstruction purposes following oral surgery is increasing, although it remains difficult to fully restore patients’ quality of life due to the resultant functional and aesthetic impairments [6]. In recent years, natural substances have increasingly been used in the treatment of various diseases, including cancer [7-9]. Such substances have both anti-inflammatory and anti-oxidant effects, and their use results in only limited toxicity and physiological activity within the human body [7,10]. Therefore, a clear need exists to develop anticancer drugs using natural substances.

    D-pinitol (3-0-methyl-D-chiro-inositol) is an active lowmolecular substance composed of a soybean oil and methyl ether compound. It has received considerable research attention due to its diverse biological activities, including its antiviral, anti-inflammatory, anti-hyperlipidemic, and anti-oxidant effects [11]. In addition, D-pinitol is structurally related to phosphatidylinositol phosphate, which participates in the insulin signaling pathway that stimulates glucose transport, and it is reported to be helpful in the treatment of diabetes [12].

    Recent studies have demonstrated the potential chemotherapeutic efficacy of lung, bladder, and breast cancer [13,14]. Further, D-pinitol has been reported to reduce the metastasis of human lung cancer [15]. However, the effects of D-pinitol on oral squamous carcinoma cell metastasis remain largely unknown.

    Metastasis is caused by uncontrolled cell proliferation, the stimulation of angiogenesis, segregation, motility, invasion into the bloodstream, and the stimulation of a new microenvironment [16]. More specifically, cancer cells separate from the primary site, migrate through the extracellular matrix, invade the bloodstream or lymphatic system, propagate distantly, and then proliferate [17]. This process is known as epithelial-mesenchymal transition (EMT), and it is caused by the reduction in adhesion between cancer cells and the increased expression of N-cadherin and vimentin during tumor progression due to the disappearance of E-cadherin, which promotes renal cell and mesenchymal transition [18]. This process is associated with various tumorigenic pathways in various types of cancers, including oral cancer [19]. The present study was designed to elucidate the effect of D-pinitol on human oral squamous carcinoma cells through apoptotic cell death, EMT, and the molecular mechanism.

    Materials and Methods

    1. Drug and reagents

    The D-pinitol used in this study was purchased from Sigma- Aldrich (St. Louis, MO, USA). It was diluted in dimethyl sulfoxide (DMSO) and then stored at –80℃. The specific antibodies for E-cadherin, N-cadherin, Snail, Slug, Caspase-3, and PARP were purchased from Cell Signaling Technology (Danvers, MA, USA), while the β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibodies of mouse anti-rabbit IgG and the rabbit anti-mouse IgG antibodies were obtained from Enzo Biochem (Farmingdale, NY, USA).

    2. Cell culture

    The human oral squamous cancer cell (OSCC) lines used in this study, namely CAL-27 and Ca9-22, were purchased from the American Tissue Culture Collection (Rockville, MD, USA). The CAL-27 and Ca9-22 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (GIBCO-BRL, Rockville, MD, USA). Both cell lines were incubated in a humidified atmosphere of 5% CO2 at 37℃.

    3. Cell viability assay

    The cell viabilities of the CAL-27 and Ca9-22 cell lines were determined by means of a 3-[4,5-dimethythiazol-2-yl]-2,5- diphenyltetrazoliumbromide (MTT) assay. Both types of cells were seeded in a 96-well plate at 1 × 10⁴ cells/well, and they were treated with different concentrations of D-pinitol (0–1.5 mM). After treatment for 24, 48, and 72 hours, the medium was removed and 0.5 mg/mL of MTT solution was added to each well and then incubated at 37℃ for 4 hours. The formazan crystals that formed were dissolved in DMSO, and the resultant solution was measured using an ELISA reader (Tecan, Mänedorf, Switzerland).

    4. Colony formation assay

    The cells were seeded in a six-well plate at 3 × 10² cells/ well, and they were treated with different concentrations of Dpinitol (0–1.5 mM) for 7 days. The colonies were fixed using 100% methanol, dyed with crystal violet for 10 minutes, and then washed using distilled water. The number of colonies was counted using an optical microscope.

    5. Hoechst 33342 staining assay

    Interms of determining the nuclear morphological change, Hoechst 33342 was used for the nuclear staining. The cells were seeded in an eight-well Lab-TekII chambered coverglass (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and they were treated with D-pinitol for 24 hours. Afterwards, the cells were fixed with 4% paraformaldehyde for 10 minutes. Next, the cells were stained with 1 μg/mL of Hoechst 33342 solution for 10 minutes in a 37℃ incubator and then washed three times with phosphate-buffered saline (PBS). The fluorescent images were observed under a Zeiss LSM700 laserscanning confocal microscope (CalZeiss, Göettingen, Germany).

    6. Wound healingassay

    The cells were cultured in a six-well plate at 1 × 106 cells and then incubated with 5% CO2 at 37℃. When the cells were 90–95% confluent, each well was scratched with pipette tip. All the wells were washed twice with PBS and treated with Dpinitol. Images were acquired after 24 hours and 48 hours, and the widths of the cell-covered areas were examined using the Image J program (version 1.41o, Java 1.6.0_10, Wayen Rasband, US National Institutes of Health, Bethesda, MD, USA).

    7. Transwell invasion assay

    A trans well with an 8.0 μm pore polycarbonate membrane (Corning Costar, Cambridge, MA, USA) was coated with 20 μL of Matrigel at 200 μg/mL and then incubated overnight. The cells were seeded (1 × 105 cells in 200 μL) and treated with 1.5 mM D-pinitol in the upper chamber of the transwell with a serum-free medium. The lower chamber was filled with 800 μL of a medium containing 10% FBS. The cells were incubated at 37℃ with 5% CO2. After 48 hours, the cells were fixed in methanol and then stained with hematoxylin and eosin for 30 minutes. The number of cells that invaded the lower chamber through the pores was counted under an inverted microscope (Olympus, Tokyo, Japan).

    8. Flow cytometry

    The cell cycle analysis of the D-pinitol-treated cells were performed by means of flow cytometry. The CAL-27 and Ca9- 22 cells were seeded in a 60 mm dish and then treated with D-pinitol for 24 hours. The cells were harvested by trypsinization and fixed with ice-cold 70% ethanol, including 0.5% Tween20, for 24 hours. The fixed cells were pelleted and washed with 1% bovine serum albumin (BSA)-PBS solution. The cells were suspended in 1 mL of PBS containing 20 μg/ mL RNaseA, incubated on ice for 30 minutes, and resuspended in a propidiumiodide (PI) solution. Next, the cell cycle was analyzed using a BD FACSverseTM flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

    9. Western blot analysis

    The cells were lysed with a 150 μL ice-cold radio immunoprecipitation assay buffer (cell signaling), which included pH7.6 50 mM Tris-Cl, 300 mM NaCl, 0.5% Triton X-100, 2 μL/ mL of a protinin, 2 mM PMSF, and 2 μL/mL of leupeptin, for 2 hours. The lysate samples were centrifuged at 13,200 rpm for 30 minutes. The protein amounts were measured using a Bradford protein assay (Bio-Rad, Richmond, CA, USA). The cell proteins were separated by means of 10% sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred on to a polyvinylidenedifluoride (PVDF) membrane. After one day, the PVDF membrane was blocked with 5% nonfat dry milk blocking buffer for 1 hour and incubated with the appropriate primary antibodies for 24 hours at 4℃. Following this, the membrane was washed with Tris-NaCl-EDTA (TNE) buffer and incubated with secondary antibodies for 2 hours at room temperature. Next, the membrane was washed again for 1 hour before SuperSignalTM WestFemto (Pierce, Rockford, IL, USA) was used for the detection of the proteins.

    10. Statistical analysis

    The results of the present study were expressed as the mean ± standard deviation. Either a Student’s t-test was used for multiple comparisons during the statistic alanalysis. The statistical analysis was performed using GraphPad PrismVersion 5.0 (GraphPad Software, San Diego, CA, USA).


    1. D-pinitol reduced the cell viability and proliferation in OSCC cells

    We performed an MTT assay to determine the cytotoxic effect of D-pinitol in CAL-27 and Ca9-22 cells. The cells were treated with various concentrations (0–1.5 mM) of D-pinitol for 24–72 hours. The cell viability was suppressed by 33% and 27% in the CAL-27 and Ca9-22 cells, respectively, when they were treated with 1.5 mM D-pinitol for 72 hours. Hence, Dpinitol reduced the cell viability in a dose- and time-dependent manner in both cell lines (Fig. 1A and 1B). To confirm the inhibitory effect of D-pinitol on cell proliferation, a colony formation assay was performed for 7 days. In both the CAL-27 and Ca9-22 cells, the number and the size of the colonies were decreased in a dose-dependent manner (Fig. 1C and 1D). These results revealed that D-pinitol attenuated the cell viability proliferative activity in OSCC cells.

    2. D-pinitol induced apoptosis in OSCC cells

    To determine whether D-pinitol induces apoptosis, we used Hoechst staining to investigate the nuclear morphological changes in CAL-27 and Ca9-22 cells that were treated with different concentrations of D-pinitol. As shown in Fig. 2A and 2B, the D-pinitol treatment caused a significant change in the treated cells’ nuclei when compared to the non-treated cells. The cells that had been treated with D-pinitol showed condensed and fragmented nuclei in a dose-dependent manner. We conducted a Western blot analysis to determine the molecules that were closely involved in the cell apoptosis. We treated the CAL-27 and Ca9-22 cells with various concentrations of D-pinitol for 24 hours. The D-pinitol activated caspase- 3 and PARP in a dose-dependent manner in both cells (Fig. 2C). Therefore, it could be concluded that the D-pinitol treatment induced apoptosis in OSCC cells.

    3. D-pinitol inhibited the cell migration and invasion in OSCC cells

    We confirmed the effect whereby D-pinitol suppressed cell migration in CAL-27 and Ca9-22 cells. The cells were treated with several concentrations of D-pinitol for 24 hours and 48 hours so that the migratory effects could be examined using a wound healing assay. As shown in Fig. 3A and 3B, the cellcovered area was reduced in a dose-dependent manner. To investigate the inhibitory effect of D-pinitol on cell invasion, a transwell invasion assay was conducted. The CAL-27 and Ca9-22 cells were treated with 1.5 mM D-pinitol for 48 hours. The D-pinitol caused a decrease in the invasive attribute of approximately 0.38-fold in the CAL-27 cells and of approximately 0.36-fold in the Ca9-22 cells when compared to the non-treated cells (Fig. 3C and 3D). These results revealed that D-pinitol inhibited the migratory and invasive properties in OSCC cells.

    4. D-pinitol regulated the EMT-related protein expression in OSCC cells

    Next, we explored the molecular mechanism by which Dpinitol suppressed the EMT in CAL-27 and Ca9-22 cells. The EMT transforms epithelial cells through the down-regulation of E-cadherin expression and the resultant mesenchymal markers, such as N-cadherin, Snail, and Slug, which promote tumor progression and metastasis in epithelial cells. As shown in Fig. 4, the D-pinitol-treated cells showed the up-regulation of Ecadherin in a dose-dependent fashion, while the N-cadherin, Snail, and Slug protein levels were all down-regulated. These results revealed that D-pinitol exerted an anti-metastatic effect on OSCC cells through the EMT process.

    5. D-pinitol induced G1 cell arrest in OSCC cells

    The arrest of the cell cycle during the G1 phase means that the cells will not be able to begin DNA synthesis and hence will remain arrested. We investigated the effect of G1 arrest in D-pinitol-treated cells using flow cytometry. In the CAL-27 and Ca9-22 cells, the flow cytometry analysis indicated that a statically significant arrest was induced during the G1 phase in a dose-dependent manner when compared to the non-treated cells, and it was accompanied by a reduction during the S and G2/M phases, too (Fig. 5A and 5B). Next, we estimated the effects of D-pinitol on the expression of the cell cycle molecular cyclin D1 and the cyclin-dependent kinase inhibitor (CKI) p21 in the CAL-27 and Ca9-22 cells using a Western blot analysis. The D-pinitol-treated cells showed the reduced expression of cyclin D1 in a dose-dependent fashion, while the p21 protein expression was induced (Fig. 5C). These results indicated that D-pinitol induced G1 arrest in OSCC cells.


    The leaves of Bougainvillea spectabilis have long been used as traditional diabetes remedies in Asia and the West Indies, and D-pinitol is a component extracted from the leaves [20]. D-pinitol is structurally related to phosphatidylinositol phosphate, which is known to participate in the insulin signaling pathway that stimulates glucose transport [21,22]. The term “diabetes mellitus” refers to a group of metabolic diseases characterized by persistent hyperglycemia, and both the prevalence and the mortality rates of these diseases are increasing. Prior investigations have reported a strong association between diabetes (especially type 2 diabetes) and carcinogenesis, which results in hyperinsulinemia, hyperglycemia, and fat-induced chronic inflammation [23]. Several studies have identified the anticancer effects of anti-diabetic drugs. For instance, metformin, a well-known drug for the treatment of type 2 diabetes, has been reported to inhibit breast, pancreas, liver, colon, ovarian, and prostate cancer [24-26]. Recently, studies have been conducted to determine whether D-pinitol has anticancer effects aside from merely lowering the blood glucose level in prostate and breast cancer cells [11,27]. However, the effect of D-pinitol on human oral squamous cell carcinoma has not yet been elucidated. Hence, in this study, we investigated the antitumor effect of D-pinitol on human oral squamous cell carcinoma CAL-27 and Ca9-22 cells.

    The induction of apoptosis is a well-known regulatory method in cancer therapy, and it is characterized by cell shrinkage, nuclear condensation, and cell cycle arrest [28]. These cell reactions are important in terms of determining the toxicity and the response to current cancer therapies because most of them target DNA [29]. It has been reported that D-pinitol induces apoptosis through the mitochondrial pathway [11] and promotes cell death via interleukin and hormones through the inhibition of nuclear factor kappa B (NF-κB) so as to mitigate tumor growth in MCF-7 cells [30]. However, no studies have previously been conducted on the cell cycle arrest caused by D-pinitol in any other cancer cells, including OSCCs. In our study, we found that D-pinitol induced nuclear condensation, which led to the fragmentation of caspase-3 and PARP, representative proteins of apoptosis, and increases in the rate of G1 arrest.

    The EMT is a crucial process for acquiring the malignant phenotype, aggression, and metastatic capacity in neoplasms. It is characterized by the loss of epithelial markers (E-cadherin) and the gain of mesenchymal markers (N-cadherin). Studies concerning the potential link with the EMT are rare in OSCCs [31]. The EMT is further characterized by increased cell migration and invasion as well as by increased resistance to apoptosis [18]. In addition, the EMT process is regulated by several transcription factors, including Snail and Slug [18,32]. Thus, the inhibition of the EMT represents a way to overcome the increased resistance on the part of apoptosis and hence to actively prevent cancer metastasis. Recently, D-pinitol has been reported to inhibit prostate cancer metastasis through inhibiting the αVβ3 integrins by modulating the FAK, c-Src, and NF-κB pathways [27], although unfortunately, save for this paper, no other studies have yet been published in this regard. In this study, we found that D-pinitol reduced the CAL-27 and Ca9-22 cell migration and invasion through the regulation of the EMT-associated marker protein expression. The Ecadherin expression increased and the N-cadherin expression decreased in a dose-dependent fashion. In addition, the Snail and Slug transcription factors decreased following treatment with D-pinitol. These results suggest that D-pinitol induces cancer resistance and inhibits metastasis, thereby showing promising potential as an anticancer agent. The results further suggest the possibility of identifying therapeutic targets for fundamental oral cancer treatment.


    This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (No. NRF-2018R1D1A1B07047739 and NRF-2019R1A2C10840 5712).



    D-pinitol reduced both the cell viability and the cell proliferation in the CAL-27 and Ca9-22 cells. (A, B) The CAL-27 and Ca9-22 cells were treated with different concentrations of D-pinitol for 24 hours. The cell viability was examined using a 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. (C, D) The cell proliferation was determined using a colony formation assay. The cells were treated with D-pinitol for 7 days and then stained with a crystal violet solution. The numbers of colonies were converted into percentages and then shown in a histogram. The result is expressed as the mean ± standard deviation.


    D-pinitol induced apoptosis in the CAL-27 and Ca9-22 cells. The cells were treated with D-pinitol for 24 hours. (A) To indicate a change in the nuclei, Hoechst staining was used in the CAL-27 and Ca9-22 cells. The cells were treated with various concentrations of D-pinitol for 24 hours. ×200. (B) The numbers of apoptosis cells were converted into percentages and then shown in a histogram. (C) To reveal the expression of those proteins closely related to cell apoptosis, such caspase-3 and PARP, a Western blot analysis was performed. β-actin was used as the loading control.


    D-pinitol reduced the cell migration and invasion in the CAL-27 and Ca9-22 cells. (A) The cells were treated with D-pinitol for 24 hours and 48 hours. (B) The fold of the initial wound area is shown in the histogram. (C) A transwell invasion assay was used to examine the invasion ability of the D-pinitol-treated cells. Hematoxlin, ×100. (D) The numbers of invasive cells were converted into percentages and then shown in a histogram.

    *p < 0.05 and **p < 0.01 for the difference between the control and the treatment groups for each group.


    D-pinitol changed the expression of the epithelial-mesenchymal transition (EMT)-related markers in the CAL-27 and Ca9-22 cells. The cells were treated with different concentrations of D-pinitol for 24 hours. The expression of the EMT-related markers, such as E-cadherin, N-cadherin, Snail, and Slug, was verified by means of a Western blot analysis. β-actin was used as the loading control.


    D-pinitol induced G1 arrest in the CAL-27 and Ca9-22 cells. Cells were treated with D-pinitol (0.1 and 0.5 mM) for 24 hours. (A) To analyze the cell cycle, the CAL-27 and Ca9-22 cells were treated different concentrations of D-pinitol and flow cytometry was performed. (B) The percentages during the G1, S, and G2/M phases were calculated and then presented in a histogram. (C) The expression of the cyclin D1 and p21 protein levels, which was closely linked to the G1 arrest, was observed by Western blot analysis. β-actin was used as the loading control.



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