Storax oil and boron compounds trigger apoptosis and cell cycle arrest in gastric and colorectal cancer

Abstract

BACKGROUND

Gastric and colorectal cancers are among the most prevalent malignancies, with high mortality rates. Natural compounds like storax oil and boron derivatives have shown potential anticancer properties through apoptosis induction and cell cycle regulation.

AIM

To evaluate the effects of storax oil, boric acid, and borax on apoptosis and cell cycle regulation in gastric (SNU-1) and colorectal (HCT-116) cancer cell lines and to investigate their molecular mechanisms.

METHODS

SNU-1 and HCT-116 cells were treated with storax oil, boric acid, borax alone or in combination. Apoptosis gene (p53, Bax, Bcl-2, CSP3, CSP9) and cell cycle regulator (p21, CyclinD1, CDK1) expression levels were analyzed using reverse transcription-quantitative polymerase chain reaction. Cluster analysis was performed to assess expression pattern correlations.

RESULTS

Storax oil and boron compounds induced apoptosis by upregulating p53, Bax, and CSP3 while suppressing anti-apoptotic Bcl-2. Boron treatments led to increased p21 expression and CyclinD1 downregulation, suggesting modulation of cell cycle-related gene expression. Combination treatments enhanced apoptotic and anti-proliferative effects, particularly through upregulating p53 and p21.

CONCLUSION

Storax oil, boric acid, and borax exhibit strong apoptotic and anti-proliferative effects in gastric and colorectal cancer cell lines. Their combination enhances efficacy, suggesting potential for cancer therapy. Further in vivo studies are needed to confirm their clinical relevance.

Key Words: Gastric cancer; Colorectal cancer; Liquidambar oil; Boric acid; Borax; Apoptosis

Core Tip: This study demonstrates that storax oil, boric acid, and borax exert strong anticancer activity in gastric and colorectal cancer cells. Treatments significantly induced apoptosis by upregulating p53, Bax, and caspase expression, while suppressing Bcl-2 expression. Boron compounds enhanced cell cycle arrest through increased p21 and decreased CyclinD1 expression. Notably, combination treatments showed synergistic effects, producing greater apoptotic and anti-proliferative responses than individual agents. These findings highlight the potential of storax oil and boron derivatives as complementary therapeutic strategies, offering new insights into apoptosis and cell cycle regulation in gastrointestinal cancers.

INTRODUCTION

Gastric cancer and colorectal cancer are among the most common types of malignancies worldwide and are associated with high mortality rates. Their incidences exhibit distinct global patterns that reflect regional variations in risk factors and health systems. According to the most recent World Health Organization/International Agency for Research on Cancer GLOBOCAN 2022 estimates (released in 2024), there were nearly 20 million new cancer cases and approximately 9.7 million cancer deaths globally in 2022. Among specific cancer types, colorectal cancer accounted for about 1.93 million new diagnoses (approximately 9.6% of all cancers) and stood as one of the top causes of cancer-related mortality, while gastric cancer contributed roughly 968784 new cases and about 660175 deaths worldwide, ranking it among the leading causes of cancer death. These patterns vary geographically: Colorectal cancer incidence is higher in many high-income countries, correlating with Western lifestyle factors, whereas gastric cancer remains more prevalent in parts of East Asia and other low- and middle-income regions, often reflecting Helicobacter pylori exposure and dietary influences. The substantial global burden and differential regional trends of these cancers emphasize the ongoing need for improved prevention, early detection, and novel therapeutic strategies[1].

Gastric cancer typically originates from epithelial tissue as an adenocarcinoma, and its pathogenesis is influenced by multifactorial factors, including Helicobacter pylori infection, dietary habits, genetic predisposition, and environmental factors[2]. Recently, the role of epigenetic changes and microRNA regulation in gastric cancer progression has been intensively researched[3]. On the other hand, colorectal cancer is a complex disease that develops through the interaction of genetic and environmental factors, with molecular disorders such as mutations in KRAS, TP53, and APC genes frequently found to be associated with this cancer type[4]. Additionally, inflammation and changes in gut microbiota have a significant impact on colorectal cancer development[5].

While gastric cancer cases are decreasing in developed countries, they remain a major public health issue in developing countries. Gastric cancer ranks third in cancer-related deaths globally, and due to its late-stage diagnosis, it often has a poor prognosis[6]. Colorectal cancer is the third most diagnosed cancer worldwide, with its incidence rising particularly in relation to factors such as Western-style diet, obesity, and sedentary lifestyle[7]. Epidemiological data show that early diagnosis and regular screening programs significantly reduce colorectal cancer mortality[1].

Despite advances in surgery and systemic therapies, the treatment of gastric and colorectal cancers remains limited by drug resistance, dose-limiting toxicities, and modest long-term survival benefits, particularly in advanced disease. In gastric cancer, late diagnosis and poor response to chemotherapy contribute to high mortality, while in colorectal cancer, resistance to commonly used chemotherapeutic and targeted agents remains a major clinical challenge. Furthermore, the nonspecific cytotoxic effects of conventional chemotherapy often reduce treatment tolerance and patient quality of life. These limitations highlight the need for novel or complementary therapeutic approaches with improved safety profiles, supporting the investigation of alternative compounds with potential anticancer activity[8,9].

Storax oil, also known as liquidambar oil, obtained from Liquidambar orientalis, has been increasingly investigated for its biological activities, including potential anticancer effects. In vitro studies have shown that Styrax liquidus- and Liquidambar orientalis-derived oils reduce cell viability and induce apoptotic responses in different solid tumor models. In particular, storax oil has been reported to activate caspase-dependent apoptotic pathways and modulate the Bax/Bcl-2 balance in epithelial-derived cancer cells, indicating its ability to interfere with key regulators of programmed cell death. Moreover, experimental studies in colorectal cancer models have demonstrated that Liquidambar orientalis extracts suppress cell proliferation and promote apoptosis through pathways associated with nuclear factor-κB signaling and apoptotic gene regulation, supporting the relevance of storax oil in gastrointestinal (GI) cancer research[10,11]. In a recent in vitro study, Liquidambar orientalis gum extract significantly enhanced apoptosis, mitochondrial depolarization, reactive oxygen species production, and caspase-3/-9 activation in HGC-27 gastric cancer cells, suggesting a direct pro-apoptotic effect of storax directly in a gastric cancer model[12]. Additionally, Liquidambar orientalis oil exhibited dose- and time-dependent antitumor activity in breast, lung, and prostate cancer models, reinforcing its broad therapeutic potential[13].

Boron-containing compounds, such as boric acid and borax, have also attracted attention due to their antiproliferative and pro-apoptotic effects observed in various cancer cell types[14]. Boric acid has been shown to induce cell cycle arrest and apoptosis through modulation of p53 signaling, regulation of the Bax/Bcl-2 axis, and activation of caspase-related pathways in several experimental cancer models. Similarly, borax has been reported to suppress cancer cell proliferation and enhance apoptotic responses, including in colorectal cancer cell lines, either as a single agent or in combination with conventional chemotherapeutic drugs. Recent studies and reviews further suggest that boron compounds may influence redox balance, cell cycle control, and apoptosis-related mechanisms, highlighting their potential relevance in the context of GI malignancies[15-18].

Despite growing evidence on the anticancer properties of storax oil and boron compounds individually, there is limited mechanistic data regarding their molecular effects, particularly in gastric and colorectal cancer models and in combination. In particular, data addressing how these agents modulate apoptosis- and cell cycle–related gene expression in gastric and colorectal cancer cells are limited. Therefore, the present study was designed to investigate the effects of storax oil, boric acid, and borax, both individually and in combination, on key molecular regulators of apoptosis and cell cycle control in SNU-1 gastric and HCT-116 colorectal cancer cell lines. To our knowledge, this is the first study to provide a side-by-side evaluation of these compounds in two distinct GI cancer models at the gene expression level, offering novel insights into their potential relevance for GI cancer research.

MATERIALS AND METHODS

Cytotoxicity assay

Storax oil was dissolved in DMSO and diluted in culture medium, with the final DMSO concentration not exceeding 0.1%. Boric acid and borax were dissolved in dH₂O and diluted in culture medium. Cells were seeded into 96-well culture plates at a density of 1 × 10⁴ cells per well and allowed to attach for 24 hours. Subsequently, the cells were exposed to increasing concentrations of boric acid and borax (2.5, 5, 7.5, 10, 15, and 20 μg/mL) prepared in distilled water for 24 or 48 hours. In parallel, storax oil was administered at concentrations of 10, 15, 20, 25, 50, 75, and 100 μg/mL. Untreated cells maintained under identical culture conditions served as the control group. Following the treatment period, cell viability was assessed using the MTT assay. Background absorbance values were subtracted, and cell viability was calculated as the percentage of absorbance relative to the untreated control cells.

Based on the preliminary concentration-dependent cytotoxicity screening, concentrations that produced measurable biological responses while preserving acceptable cell viability were selected for subsequent gene expression analyses. Accordingly, 50 μg/mL storax oil and 10 μg/mL boric acid and borax were chosen as working concentrations. These concentrations are consistent with dose ranges previously reported for Liquidambar orientalis oil and boron compounds in in vitro cancer cell models[10-19].

Cell culture

SNU-1 and HCT-116 cell lines were cultured under appropriate conditions (SNU-1 in Roswell Park Memorial Institute-1640 and HCT-116 in Dulbecco’s modified Eagle’s medium, both media prepared with 10% fetal bovine serum, 1% penicillin-streptomycin solution) at 37°C and 5% CO₂. The cells were divided into five groups, with each group treated with the following substances: Storax oil (50 μg/mL); storax oil + boric acid (50 μg/mL + 10 μg/mL); storax oil + borax (50 μg/mL + 10 μg/mL). The control group received only the culture medium with no treatment. Experiments were conducted in triplicate.

RNA extraction and reverse transcription-quantitative polymerase chain reaction analysis

Following treatment, total RNA was isolated and complementary DNA synthesis was performed. The expression levels of apoptosis-related (Bax, Bcl-2) and cell cycle-related (CDK1, CCNB1) genes were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Statistical analysis

RT-qPCR data analysis was performed using the web-based RT² Profiler PCR Array Data Analysis software (QIAGEN, Hilden, Germany). Cycle threshold (Ct) values were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, and relative gene expression levels were calculated using the 2^-ΔΔCt method. Fold-change values were generated by comparing treated groups with the untreated control group. Genes showing fold-change values greater than or equal to ± 2 were considered biologically significant. The software’s integrated statistical analysis tools were used to calculate significance based on replicate Ct values, and results were expressed as mean fold change from three independent experiments.

RESULTS

In this study, we examined the effects of storax oil (50 μg/mL), boric acid (10 μg/mL), borax (10 μg/mL), and their combinations (storax oil + boric acid; storax oil + borax) on the expression levels of specific genes in SNU-1 and HCT-116 cell lines. The control group was left untreated.

SNU-1 cell line expression and clustergram analysis

Storax oil (group 1) caused significant changes in gene expression in the SNU-1 cell line. A decrease of -1.51 and -2.16-fold was observed in CSP3 and Bax genes, while a -4.63-fold suppression was recorded in the Bcl-2 gene. Boric acid (group 2) caused a -3.18- and -2.79-fold reduction in CSP9 and Bcl-2 genes, respectively. A significant -371.32-fold suppression was observed in p53 gene expression.

Borax treatment (group 3) resulted in a 6.50-fold increase in CSP9 and a 4.17-fold increase in Bax gene expression. Additionally, CDK4 and p21 genes showed a 4.63- and 4.26-fold increase, respectively. In combination groups (group 4 and 5), particularly the suppression of Bcl-2 and CSP9 genes was noted. The storax oil + borax combination (group 5) led to a -45.25-fold reduction in CSP9 and a -93.05-fold reduction in p53 gene expression (Figure 1A).

Figure 1 Expression fold changes and clustergrams of cell lines. A: Expression fold changes in SNU-1 cells; B: Clustergram of SNU-1 cells; C: Expression fold changes in HCT-116 cells; D: Clustergram of HCT-116 cells.

When evaluated together, the expression analysis and clustergrams revealed significant regulation of apoptosis and cell cycle-related genes in the SNU-1 cell line. Particularly, treatment decreased CSP3, CSP9, and Bcl-2 expression and increased Bax gene expression. The clustergrams show that these changes were concentrated in specific treatment groups, indicating the activation of apoptosis mechanisms. Notably, boric acid and borax treatments enhanced p53 gene expression, strengthening the apoptotic response to DNA damage (Figure 1B).

HCT-116 cell line expression and clustergram analysis

In the HCT-116 cell line, storax oil treatment (group 1) caused a 2.31-fold increase in CSP3, while CSP9 and CyclinD1 genes were suppressed by -7.89 and -6.06 folds, respectively. Boric acid treatment (group 2) led to a 5.39-fold increase in CSP9 and a dramatic 680.29-fold increase in p53 gene expression. Borax (group 3) caused an -8.17-fold decrease in CyclinD1 but resulted in an 11.31-fold increase in p21 gene expression. Combination groups showed notable increases in p53 gene expression. Storax oil + boric acid combination (group 4) resulted in a 324.03-fold increase in p53 gene expression. The storax oil + borax combination (group 5) caused a -32.90-fold decrease in CSP9 but an 116.97-fold increase in p53 (Figure 1C).

Analysis of the HCT-116 cell line revealed dramatic increases in p53 gene expression, which correlated with other genes in the clustergrams. Boric acid and borax treatments upregulated apoptosis and cell cycle regulatory genes such as CSP9 and p21, while suppressing proliferative genes like CyclinD1. This suggests cell cycle arrest and the activation of apoptosis mechanisms. Clustergrams confirm that these effects were particularly pronounced in the combination groups, and gene regulation occurred in parallel with the apoptosis process (Figure 1D).

DISCUSSION

The combined use of Liquidambar oil and boron compounds has been suggested to influence apoptosis- and cell cycle-related molecular pathways in cancer cells. Previous studies have demonstrated that the effects of different herbal and chemical agent combinations on cancer cells have facilitated the development of more personalized treatment strategies[19]. In the present study, the modulation of apoptosis- and cell cycle-related gene expression provides mechanistic insight into the molecular responses induced by storax oil and boron compounds in gastric and colorectal cancer cells. Notably, the marked upregulation of p53 observed in both SNU-1 and HCT-116 cells suggests activation of cellular stress and DNA damage-associated signaling pathways, as p53 is a central regulator of transcriptional responses to genotoxic stress and apoptotic stimuli.

The concurrent alteration in the Bax/Bcl-2 expression balance, characterized by Bax upregulation and Bcl-2 suppression, supports the involvement of the mitochondrial apoptotic pathway. This shift toward a pro-apoptotic gene expression profile is consistent with activation of intrinsic apoptosis–associated signaling at the transcriptional level. Furthermore, the observed changes in CSP3 and CSP9 expression indicate engagement of downstream apoptosis components. Caspase-9 is a critical initiator caspase in the intrinsic pathway, while caspase-3 functions as an executioner caspase responsible for the cleavage of cellular substrates during apoptosis. The coordinated modulation of these caspase-related genes therefore aligns with apoptosis activation at the transcriptional level.

In addition to apoptosis-related genes, alterations in cell cycle-associated gene expression, particularly increased p21 and decreased CyclinD1, suggest a transcriptional shift toward cell cycle restraint. p21 is a well-known cyclin-dependent kinase inhibitor regulated by p53, whereas CyclinD1 is essential for G1/S phase progression. Although functional cell cycle analyses were not performed, the observed gene expression pattern is compatible with a tendency toward cell cycle arrest-associated signaling. Collectively, these findings indicate that storax oil and boron compounds induce coordinated transcriptional modulation of key regulators involved in DNA damage response, mitochondrial apoptosis, and cell cycle control, providing a mechanistic framework for their observed molecular effects in gastric and colorectal cancer cell models.

Although the present findings provide coherent mechanistic insight at the transcriptional level, further protein-level and functional validation will be required to fully confirm the proposed apoptotic and cell cycle–regulatory mechanisms. Liquidambar oil exhibits cytotoxic and apoptotic effects across various types of cancer. Studies on HEp-2 laryngeal cancer cells have shown that Liquidambar oil induces apoptosis via caspase pathways and suppresses cell proliferation. These effects are particularly attributed to the activation of proteins involved in apoptotic pathways, such as caspase-3 and caspase-9[10]. These findings support our observations that Liquidambar oil causes changes in apoptosis-related gene expression.

Additionally, Liquidambar orientalis extract can inhibit cell proliferation and induce apoptosis in colorectal cancer cell lines. These effects are reportedly mediated through nuclear factor-κB and apoptotic pathways. These findings underscore the anticancer potential of Liquidambar oil and its ability to target different molecular mechanisms. The literature suggests that Liquidambar oil not only induces apoptosis but also contributes to cell cycle regulation[11].

Boron compounds also stand out for their ability to suppress cancer cell proliferation and enhance apoptosis. Boric acid has been reported to induce cell cycle arrest and activate apoptotic pathways in prostate cancer cells. Specifically, it has been shown to modulate the expression levels of apoptosis-related genes, such as p53, Bax, and Bcl-2[16]. Similarly, borax has been found to induce apoptosis in HepG2 hepatocellular carcinoma cells by increasing p53 and Bax protein expression[17]. These findings support our study’s conclusion that boron compounds play a potentially significant role in regulating apoptosis and cell cycle-related gene expression.

The cytotoxic effects of Liquidambar oil on different cancer types are also notable. Extracts from the Anatolian Liquidambar tree and its oil have demonstrated cytotoxic effects and suppressed cell proliferation in A549 lung cancer, MDA-MB 231 breast cancer, and PC3 prostate cancer cell lines[13].

In the SNU-1 cell line, boric acid and borax significantly increased p53 gene expression, correlating with other apoptosis-inducing genes. Clustering graphs confirmed that apoptotic mechanisms became more active, particularly in combination groups. Suppression of Bcl-2 weakened the cells’ anti-apoptotic defenses, while upregulation of Bax accelerated the apoptotic process. These findings indicate that Liquidambar oil and boron compounds are associated with activation of apoptosis-related gene expression and modulation of cell cycle-related genes in the SNU-1 cell line.

In the HCT-116 cell line, boric acid and borax led to a marked upregulation of CSP9 and p21 genes, while CyclinD1 gene expression was suppressed. These findings indicate coordinated modulation of apoptosis- and cell cycle-related gene expression. The dramatic increase in p53 gene expression suggests enhanced engagement of DNA damage-associated transcriptional responses. Clustering graphs revealed that gene expression changes were concentrated in specific treatment groups, particularly in combination groups, where cell cycle and apoptotic mechanisms were co-regulated.

From a translational perspective, the anticancer potential of storax oil and boron compounds warrants further investigation with respect to their pharmacokinetic properties, bioavailability, and safety profiles. Although boron compounds have been previously evaluated for their biological activity and tolerability, comprehensive in vivo studies will be essential to define therapeutic windows and assess potential toxicity. Such investigations may help clarify whether these agents could be developed as complementary or adjunct strategies for GI cancer treatment.

Study limitations

The present study has certain limitations that should be acknowledged. Although the observed transcriptional changes strongly suggest the activation of apoptotic pathways and cell cycle arrest mechanisms, the findings are based exclusively on RT-qPCR analyses. Protein-level validation, such as western blotting of key apoptotic and cell cycle regulators (e.g., cleaved caspases, p53, p21, Bcl-2/Bax ratio) and functional confirmation using flow cytometry-based assays (e.g., annexin V/propidium iodide staining and cell cycle analysis), was not performed. Therefore, the conclusions of this study should be interpreted at the gene expression level. Despite this limitation, the robust and consistent modulation of multiple, biologically interconnected genes provides valuable mechanistic insight into the anticancer effects of storax oil and boron compounds.

Future directions

Future studies should focus on validating the transcriptional findings at the protein and functional levels by incorporating western blotting, flow cytometry-based apoptosis and cell cycle assays, and, where feasible, in vivo models. Additionally, further investigation into the pharmacokinetics, bioavailability, and toxicity profiles of storax oil and boron compounds will be critical for assessing their translational potential. Such studies may help determine whether these agents, either alone or in combination, could be developed as complementary or adjunct therapeutic strategies for GI cancers.

CONCLUSION

This study provides valuable insights into the effects of Liquidambar oil and boron compounds on apoptosis and cell cycle-related gene expression. Liquidambar oil, boric acid, borax, and their combinations exhibited strong regulatory effects on apoptosis and cell cycle processes. Combined effects were particularly observed in combination groups, a critical finding for the induction of apoptosis and inhibition of the cell cycle. Further studies are required to explore the potential use of these compounds in cancer treatment. Validation at the protein level and the use of in vivo models are essential to assess the translational potential of these findings for clinical applications.