Integrated Metabolomics and Proteomics Analyses to Reveal Anticancer Mechanism of Hemp Oil Extract in Colorectal Cancer

doi:10.21203/rs.3.rs-3988139/v1

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Research Article

Integrated Metabolomics and Proteomics Analyses to Reveal Anticancer Mechanism of Hemp Oil Extract in Colorectal Cancer

https://doi.org/10.21203/rs.3.rs-3988139/v1

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published 01 Jul, 2024

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Background

Cannabis sativa L. has a lengthy medical history in Chinese folk medicine. Industrial cannabis, also known as hemp, refers to cannabis strains with no addictive effect and holds great economic and medical value. Hemp exhibited multiple pharmaceutical activities with anti-inflammatory, antioxidant, and anti-tumor effects. Hemp oil extract (HOE), a mixture of cannabinoid-rich components extracted from hemp, has shown potential in treating colorectal cancer (CRC). However, the specific anticancer mechanisms of HOE remain unclear. This research aims to elucidate the anticancer mechanisms of HOE in the treatment of CRC by employing an integrated omic approach, analyzing changes in cellular metabolites and proteins.

Methods

In this study, we employed mass spectrometry-based omic approaches, specifically metabolomics and proteomics, to comprehensively investigate the global effects of HOE on colorectal cancer cells. Bioinformatics analysis including bulk RNA-seq and single-cell RNA-seq were utilized to unravel specific gene expression differences and heterogeneity in CRC. The inferred conclusions were confirmed by utilizing flow cytometry, western blot, and immunohistochemistry techniques.

Results

The in vitro and in vivo experiments indicated that HOE induced significant changes in purine metabolism pathways, down-regulated c-MYC and inhibited the expression of cell cycle-related proteins, including CCND1, CDK4, CDK6, which herein arrested cell cycle in G1 phase.

Conclusion

Through Integrating Metabolomics and Proteomics approaches, a comprehensive analysis of anticancer mechanisms of HOE on CRC was conducted. The study demonstrated that HOE blocks the cell cycle in the G1 phase by inhibiting c-MYC, leading to the inhibition of colorectal cancer cell proliferation. These findings provide experimental evidence for the potential use of hemp in medicine.

Hemp oil extract

Colorectal cancer

Systems biology approach

Cell cycle arrest

HOE effectively suppressed CRC cell proliferation both in vitro and in vivo.

HOE induced cell cycle arrest at the G1 phase by targeting and inhibiting the c-MYC protein.

Revealed comprehensive changes in proteome and metabolome of colorectal cancer cells following HOE intervention.

Cannabis sativa L. is a plant in the Cannabaceae family with a long history of medical applications in Chinese folk medicine dating back around 2700 BC[1, 2]. Despite its potential benefits, its psycho-addictive component, tetrahydrocannabinol (THC), limits its use. However, industrial Cannabis, or hemp, contains less than 0.3% THC and is used in various industries[3]. Some countries have legalized or partially legalized hemp due to its medical value[4], including its anti-inflammatory, antioxidant, and anti-tumor effects. Hemp has been applied in military medicine to alleviate health problems such as post-traumatic stress disorder, substance use disorder, sleep disturbance, and chronic pain[5–7]. Some states in America have approved medical uses of hemp, including for veterans' health problems[8]. However, research on its composition, pharmacology, and even toxicity is still lagging behind its widespread application, despite its prevalence.

Cannabinoids are the active compounds in hemp, with over 100 compounds in addition to THC[9, 10]. Some cannabinoids have been approved for medical use, such as dronabinol was utilized in the therapy of weight loss in Acquired Immune Deficiency Syndrome (AIDS) patients and cannabidiol was approved by the Food and Drug Administration for severe epileptic conditions[11]. Studies have shown that some cannabinoids (such as cannabidiol, cannabidivarin, and cannabidiolic acid) have anti-tumor effects by promoting cancer cell apoptosis and ROS production[12]. Throughout history, cannabis extracts were commonly utilized for medicinal purposes[13], and it is plausible that its components exhibited synergistic effects. Hemp oil extract (HOE) is a mixture of cannabinoid-rich components extracted from the flowers and leaves of industrial hemp. However, the majority of experiments simply elaborated on the mechanism of a single cannabinoid or a few cannabinoids. The specific anticancer mechanism of the total cannabinoid components is still unclear and remains to be discovered.

Cases have demonstrated a possible benefit of HOE intake that may have resulted in the observed tumor regression[14]. In the present study, HOE has shown potential in treating colorectal cancer (CRC), which is a common and deadly malignancy worldwide. Conventional CRC treatment has limitations, such as side effects and resistance to chemotherapy, leading to the search for alternative treatments such as natural products[15, 16]. Medications produced from natural plants may have lower toxicity compared to current drugs and can target multiple treatment sites, making them a more promising source of medication with greater potential. One possible reason for the observed tumor regression with HOE intake is that HOE has multiple ingredients and complex targets, which may collectively contribute to its anti-tumor effects. However, traditional analytical methods are inadequate for comprehensively evaluating the complex multi-component and multi-target mechanism of HOE, due to that one single research method hardly clarifies the complex mechanism or explores the full process of HOE treatment. Systems biology methods, such as transcriptomics, proteomics and metabolomics can be used to explore the interactions between multiple substances in biological systems and discover the regularity of changes from a systemic perspective. In this study, we investigated the substance basis of HOE and explored its mechanism of action against CRC using a multi-omics approach. Our study identified the chemical composition of HOE and validated its anti-tumor activities in vitro and in vivo. By depicting the alterations in protein and metabolic profiles of colorectal cancer cells after HOE intervention, we further revealed that HOE exerts anti-CRC effects by inhibiting the cell cycle through inhibiting c-MYC. Our findings not only highlight the therapeutic potential of hemp but also establish a framework for exploring new mechanisms of action in other natural plants.

2.1. Antibodies and reagents

The primary antibody against c-MYC (BF8036) was purchased from Affinity Biosciences (Jiangsu, China). Primary antibodies against α-Tubulin (AC012), CCND1 (A11022), CDK4 (A0366), and CDK6 (A1545) were purchased from ABclonal (Wuhan, China). The secondary antibodies conjugated to HRP were obtained from Proteintech (Wuhan, China). HOE was purchased from Hansu Biotechnology Company (Yunnan, China). Cell counting kit-8 (CCK-8) reagent, ultra ECL Western blotting detection reagent were purchased from Yeasen Biotechnology (Shanghai, China). 4% paraformaldehyde fix solution, crystal violet, BCA protein assay kit, dimethyl sulfoxide (DMSO), cell cycle and apoptosis analysis kit were purchased from Beyotime Biotechnology (Shanghai, China). Acetonitrile (ACN), methanol, formic acid, 2-chloroacetamide (CAA), triethylammonium bicarbonate (TEAB), tris (2-carboxyethyl) phosphine (TCEP), trifluoroacetic acid (TFA), ammonium formate, and tris-(hydroxymethyl) aminomethane (Tris), protease and phosphatase inhibitor cocktail were purchased from Sigma-Aldrich (the United States). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), pierce quantitative colorimetric peptide assay kit, and tandem mass tags (TMT, Catalog NO 1863006) were purchased from Thermo Fisher Scientific (Shanghai, China).

2.2. Chemical profiling of HOE

The HOE stock solution was prepared as follows: 10 mg HOE was ultrasonicated with 1 mL methanol, then frozen at -80°C for further use. The chemical profiling of HOE was performed using a UPLC system (Waters Technologies Co., Ltd., Milford, MA, United States) coupled with a Triple TOF 5600 plus mass spectrometer (AB SCIEX, Framingham, MA, United States). HOE stock solution was diluted to 0.1 mg/mL and the injection volume was set as 1 µL. The liquid chromatographic separation was carried out on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters, Milford, MA, USA) at a flow rate of 0.3 mL/min at 35°C. The optimal mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient elution as follows: 0–23 min, 30–95% B; 23–26 min, 95% B. The MS detection parameter and data processing software were the same as the method reported by Yi et al.[17]. The gas chromatographic separation was carried out on an HP-5MS capillary column (0.25 mm in diameter × 30 m in length and 0.25 µm film thickness, Agilent Technologies, Santa Clara, CA, USA). The injector temperature was set at 250°C and the split ratio was 5:1.The initial column temperature was set at 50°C and maintained at the rate of an increase of 10°C/min until 200°C and then held for 30 min. Pure helium gas (99.99%) was used as the carrier gas with a flow rate of 1.0 mL/min and the electron ionization voltage was 70 eV. Data were obtained continuously by collecting the full-scan mode mass spectrum within the scan range from 50 to 500 (m/z).

Structures were tentatively identified based on the exact molecular weight and MS/MS spectra by referring to the chemical information with those in the literature and Reaxys (https://www.reaxys.com/) database(for UPLC-MS data searching) and NIST standard reference database (for GC-MS data searching).

2.3. Cell cultures and cell viability assay

The SW620 cells were cultured in DMEM supplemented with 10% FBS in a biochemical incubator with a humidified atmosphere containing 5% CO₂ at 37°C. The logarithmic phase cells were selected for experimentation and were seeded into a 96-well plate with 5000 cells in each well. Three groups were set up, including the blank group (medium only), the control group (cells with medium) and the treatment groups (treated with HOE at 3 and 6 µg/mL, respectively). After being treated with HOE for 24 h, 48 h, and 72 h, 10 µL of CCK-8 reagents was added to each well and cultured for another 1 h. The absorbance (A) at 450 nm wavelength was measured and the cell viability was calculated as [(A_sample−A_blank) / (A_control−A_blank)] × 100%, where A_sample, A_control, and A_blank stands for the absorbance of the treatment group, the control group, and the blank group, respectively.

2.4. Colony-formation assay

SW620 cells were seeded into 6-well plates containing medium supplemented with the designated concentrations of HOE (0, 3, 6 µg/mL) and cultured for 2 weeks. After 2 weeks, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. The visible colonies were photographed using a digital camera.

2.5. Animal studies

The male BALB/c nude mice were purchased from Hangzhou Medical College. The SW620 cells were cultured in DMEM supplemented with 10% FBS in a biochemical incubator with a humidified atmosphere containing 5% CO₂ at 37°C.

After being maintained for stableness, the cells at 2×10⁶ cells density were aseptically injected into the subcutaneous right forelimb of mice for tumorigenesis. When xenograft tumors were visible, mice were randomly assigned into two groups (Control group and HOE group, n = 6 in each group). Mice in the HOE group were intraperitoneally injected with 30 mg/kg HOE every 3 days, while the mice in the control group were given an equal amount of normal saline at the same time points. Tumor dimensions and mice weight were monitored every 3 days, and tumor volumes were calculated with V = (Length × Width²)/2. On day 15, mice were sacrificed, and tumors were resected for further experiments. The Laboratory Animal Welfare and Ethics Committee of Zhejiang University approved the protocols for animal studies (AP CODE: ZJU20220457). All experiments conformed to all relevant regulatory standards.

2.6. H&E staining

The mouse tumor specimens were collected and fixed in 10% neutral-buffered formalin and then routinely paraffin-embedded. The paraffin sections (3 µm thickness) were affixed to micro-slides (MUTO-GLASS, Japan) and air-dried overnight at 37°C. Then, the sections were dewaxed in xylene and rehydrated through a descending-graded alcohol series. Finally, the sections were subjected to hematoxylin and eosin (H&E) staining.

2.7. Immunohistochemistry (IHC) staining

Formalin-fixed and paraffin-embedded sections were prepared from the xenograft tumors to perform an immunohistochemistry (IHC) assay. All 3-µm-thick sections of tumor samples were used for dewax and rehydration via different concentrations of xylene and ethanol. After eradicating endogenous peroxidase activity with 3% H₂O₂ and blocking non-specific antigens with 5% bovine serum albumin (BSA) buffer, the sections were subjected to incubation with primary antibodies of CCND1, c-MYC, CDK6, and CDK4 (dilution fold = 1:100) at 4℃ overnight. After washing with phosphate buffer saline (PBS) several times, the sections were further incubated with the secondary antibody of rabbit anti-mouse IgG for 50 min at room temperature. The section signals were measured using a DAB substrate working solution. The nuclei were stained with hematoxylin dye, and then the positive cells in each section were photographed through an inverted fluorescence microscope (BX60, Olympus, Japan).

2.8. TMT-labeled quantitative proteomic analysis

The SW620 cells were seeded into a 6-well plate for 24 h and then treated with HOE at concentration of 3 µg/mL, with three biological replications in each group. After 24 h of culture, the protein was extracted, reduced, alkylated, and digested into peptide. Then the peptide was labeled and fractionated. Subsequently, an amount of 2 µg peptide collected from each fraction was subjected to Proxeon 1000 UHPLC system coupled with an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) and separated on a 50 cm × 75 µm Easy Spray column (Thermo Scientific). The raw MS data were acquired by data-dependent acquisition at a resolution of 75,000 and 50,000 in MS and MS/MS, respectively. The protein search and quantification were generated in Proteome Discoverer 2.1 software (Thermo Scientific), and Mascot 2.6.0 (Matrix Science). The specific methods and parameters referred to the previous study[18].

2.9. Untargeted metabolomics analysis

The SW620 cells were seeded into a 6-well plate for 24 h and then treated with HOE at concentration of 3 µg/mL, with six parallel wells in each group. After 48 h of culture, the cells in each well were harvested with 80% methanol, frozen and thawed three times with liquid nitrogen, and centrifuged at 20,000 rpm for 20 minutes at 4°C. The protein concentration was measured using a BCA assay to normalize re-solubilization volume, and the supernatant was freeze-dried for further analysis via low-temperature centrifugation. The quality control (QC) samples were created by combining 5 µL solution from all samples. The specific methods and parameters referred to the previous study[19]. R was used for multivariate statistical analysis, visualization of the data, orthogonal partial least squares-discriminant analysis (OPLS-DA), volcano plot, diagram, and heat map. The functional enrichment analysis was performed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) and the ChemRICH enrichment was processed using ChemRICH (http://chemrich.fiehnlab.ucdavis.edu/).

2.10. Flow cytometric analysis

The SW620 cells were seeded into a 6-well plate for 24 h and then treated with HOE at concentration of 3 µg/mL, with three parallel wells in each group. After 24 h or 48 h of culture, the cells were stained with propidium iodide and analyzed using flow cytometry with 488 nm excitation wavelength to measure red fluorescence and light scattering.

2.11. Western blotting

The cells were seeded in 6-well plates with three biological replicates and treated with 0, 3, or 6 µg/mL for 24 h. Afterward, the cells were washed with 1 mL ice-cold PBS and lysed with 200 µL ice-cold lysis buffer containing a protease inhibitor. The adherent cells were collected using a cold plastic cell scraper, transferred into a pre-cooled microcentrifuge tube, and centrifuged at 4°C for 20 minutes at 12,000 g to obtain the supernatant. The protein concentration of the cell lysates was measured by using a BCA Protein Assay Kit. 50 µg of protein was separated on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-rad, United States) for immunoblotting. The membranes were blocked with 5% non-fat milk powder at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C, including anti-c-MYC (1:1000), anti-CCND1 (1:1000), anti-CDK4(1:1000), anti-CDK6(1:1000), and α-Tubulin antibody (1:4000). On the following day, the membranes were incubated with fluorescent labeling HRP-conjugated Affinipure Rabbit Anti-Mouse IgG (H + L) (1:10000) for 2 h at room temperature. Finally, the protein bands were visualized using the ultra ECL western blotting detection reagent and detected by the ChemiDoc MP Imaging System (Bio-rad, United States). The band intensities were quantified using ImageJ software, and the protein expression was normalized to α-Tubulin.

2.12. Single-cell RNA-seq and data processing

Single-cell RNA-seq data of 62 CRC and 36 adjacent normal colon tissues was collected from the Single Cell Portal (https://singlecell.broadinstitute.org/single_cell) [20], the data processing including data loading, quality control, normalization, integration, clustering, differential expression and expression visualization was performed on this site.

2.13. Data statistics and analysis

All results were presented as mean ± standard error of the mean (SEM). Data analysis and graphics were produced using the R toolkit and statistical analysis was conducted by using the Student’s test and one-way analysis of variance (ANOVA).

3.1. Chemical profiling of HOE

In this current investigation, we employed a comprehensive analytical approach utilizing UPLC-MS coupled with GC-MS to delineate the specific components within HOE. The mass spectra of both GC (Fig. 1A) and UPLC (Fig. 1B-C) are presented in Fig. 1. Through a meticulous comparison of mass spectrum data and consideration of physicochemical properties of known compounds, we successfully identified 13 compounds through UPLC-MS and 6 compounds through GC-MS. Ultimately, a total of 16 representative compounds, including cannabidiol, cannabidivarin, cannabinol, among others, were tentatively identified. This robust identification establishes a foundational framework for subsequent research endeavors.

3.2. HOE inhibits CRC cell proliferation in vitro and in vivo

To investigate the impact of HOE on CRC cell proliferation, SW620 cells were treated with 3 and 6 µg/mL of HOE for 24, 48 and 72 h. The results showed that the cell viability of SW620 decreased in a time and dose-dependent manner (Fig. 2A-C). In comparison to the normal colon primary cell line, HCoEpic, HOE exhibited a greater inhibitory effect in various CRC cells such as HCT-116, SW620, SW480, and HT-29 (Fig. 2D), indicating that HOE has a general inhibitory effect on CRC cell growth while causing minimal damage to normal cells. As depicted in Fig. 2E, HOE suppressed colony formation in SW620 cells after two weeks of treatment, with higher concentrations yielding higher inhibition rates.

To assess the potential effects of HOE on CRC in vivo, a xenograft model using the SW620 cell line was established in nude mice(Fig. 2F). As shown in Fig. 2G, HOE induced a significant reduction in tumor volume, the body weight and tumor volume of mice were measured every three days, and the results demonstrated that HOE could inhibit the tumor growth while not significantly affecting the mice’s weight (Fig. 2H-I). H&E staining revealed that the number of tumor cells in the model group is greater than in the HOE treatment group (Fig. 2J-K). Additionally, the protein levels of the proliferation marker Ki-67 were determined by IHC staining (Fig. 2L-M), which showed that the treatment with HOE decreased Ki-67 expression. These findings indicate that HOE suppresses tumor cell proliferation in the CRC xenograft model. Overall, these observations demonstrate that HOE can inhibit CRC proliferation in vitro and in vivo with minimal side effects.

3.3. Quantitative proteomics-coupled with bioinformatics displays the landscape of HOE-regulated protein networks

To validate and reveal the potential mechanism of HOE in treating of CRC, the quantitative proteomics was conducted in comparison of the HOE treatment group and the vehicle group. The protein was initially extracted from the cells and subsequently hydrolyzed into peptides, which were subsequently labeled with TMT agents. Following mixing and fractionation, the resultant samples were subsequently introduced into the UHPLC-MS/MS system (Fig. 3A). A total of 5853 proteins, including 134 up-regulated proteins and 139 down-regulated proteins, were identified as the differentially expressed proteins (DEPs). The volcano plot was presented based on the threshold value of |Fold change| ≥ 1.2 and p-value < 0.05. Figure 3B encapsulates the dysregulated proteins inclusive of CDK4, PLK1, CCNB2, c-MYC, exhibiting downregulation, and APOB, GPAT3, depicting upregulation. Accordingly, the protein-protein interaction network of the DEPs was generated, with c-MYC exhibiting the highest degree value as one of the significant hubs of the network (Fig. 3C), indicating the crucial role of c-MYC in the anti-tumor effects of HOE. To further investigate the potential mechanism, KEGG functional enrichment results of these dysregulated proteins are presented (Fig. 3D), with the top 15 signaling pathways enriched including cell cycle, DNA replication, pyrimidine metabolism, purine metabolism, etc.. And cluster and enrichment analysis was conducted with Metascape. The DEPs were colored by cluster ID, where nodes that share the same cluster ID are typically close to each other and belong to the same pathway, which including cell cycle, DNA metabolic process, mitotic cell cycle, etc. (Fig. 3E). Then, we used DAVID to perform the GO analysis to reveal the functions of DEPs. The significant GO terms and major proteins were shown in the chord plot, which demonstrating that the functions of DEPs are tightly linked to the cell cycle pathway. Overall, the results of the quantitative proteomic confirmed that HOE could exhibit anti-tumor effects by affecting the cell cycle pathway.

3.4. Metabolomics reveals the profiling of HOE-regulated metabolites

Metabolomics analysis was performed to explore the metabolic alterations upon HOE (3 ug/mL) treatment. In order to delineate differential metabolite profiles between the control and HOE groups, OPLS-DA models were meticulously constructed. In this context, the established OPLS-DA models (Fig. 4A-B) exhibited a commendable fit and impressive predictability.

Next, for further refinement in the selection of differential metabolites, the volcano plot was presented to depict differential metabolites after HOE treatment in the positive (C) and negative (D) modes, respectively(Fig. 4C-D). Based on the threshold value of fold change (≤ 0.83 or ≥ 1.2) and p-value (< 0.05), 1056 differential ions in positive mode and 1915 differential ions in negative mode were acquired. 53 metabolites showing significant differences in the HOE treatment group were identified, which were markedly differentiable from the control group, whose relative levels between the two groups were significantly different demonstrated in Heatmap determined by LC-MS, normalized by Z-score (Fig. 4E). Of significant note, a marked decrease was observed in the levels of crucial metabolites, including ATP (FC = 0.28, p = 0.001), GTP (FC = 0.22, p = 0.003), adenine (FC = 0.25, p = 0.001), and Guanine (FC = 0.72, p = 0.015), within the purine metabolic pathway. Conversely, a significant increase was observed in the concentration of GMP (FC = 1.34, p = 0.004), xanthosine (FC = 5.04, p = 0.001), and xanthine (FC = 4.36, p = 0.001).

Functional enrichment analysis, grounded in the altered metabolites, revealed the top 11 distinctive metabolic pathways differentiating the two groups, as depicted in Fig. 4F. These pathways encompassed purine metabolism, pyrimidine metabolism, nucleotide sugars metabolism, among others. In summary, the pathways of purine and pyrimidine metabolism underwent significant alterations following HOE treatment, primarily exhibiting a discernible decreasing trend.

Given the limitations of functional enrichment analysis relying on sparse biochemical knowledge annotations [21], we sought to delve deeper into the altered metabolites from the perspective of chemical structure. For this purpose, we employed ChemRICH analysis, a statistical enrichment approach that focuses on chemical similarity to cluster metabolites with similar structures. ChemRICH statistics revealed significant differences in purine nucleosides, specifically adenine and guanine nucleotides, and pyrimidine nucleosides (Fig. 4G). Simultaneously, a Tanimoto chemical similarity clustering tree was constructed to visually represent the clustering of metabolites with distinct structures, as illustrated in Fig. 4H. Figure 4I illustrates the specific relative levels of metabolites associated with purine metabolism, all of which exhibited significant differences between the HOE-treated and control groups.

In summary, our findings suggest that HOE disrupted the purine and pyrimidine metabolism in CRC cells. As these endogenous substances are essential for cell cycle progression [22], the metabolome results are consistent with the proteome. This comprehensive analysis, combining functional enrichment and chemical structure perspectives, enhances our understanding of the intricate metabolic alterations induced by HOE treatment.

3.5. HOE arrests cells in the G1 phase by inhibiting c-MYC

Based on the result of integrated proteomics and metabolomics method, we hypothesized that HOE functioned as an anti-CRC agent by arresting cell cycle via c-MYC, leading to the disruption of purine and pyrimidine metabolism. Flow cytometric analysis showed that G1-phase cells increased from 46.5–61.9% after treatment of HOE on SW620 cells for 24 h (Fig. 5A-B), which indicated that HOE arrested the cell cycle at the G1 phase in SW620 cells. To further confirm the mechanism of HOE on the cell cycle of the SW620 cell line, we examined the protein expression of c-MYC and other cycle-related proteins by western blotting (Fig. 5C-D). The results showed that HOE dose-dependently decreased the protein expression of c-MYC, CCND1, CDK4, and CDK6. Using the xenograft mouse model, we measured the levels of these four proteins by means of immunohistochemistry staining, whose results were consistent with the cellular level demonstrating exposure to HOE occurred with varied degrees of decrease (Fig. 5E-F). Therefore, HOE arrested the cell cycle of SW620 by modulating the level of c-MYC to realize the inhibition of carcinogenesis and progression of CRC.

3.6. Bioinformatics analysis unraveled specific gene expression differences and heterogeneity in CRC

To assess the potential clinical applicability of HOE, we comprehensively evaluated the expression profiles of the four critical genes, i.e., c-MYC, CCND1, CDK4, and CDK6, in clinical samples of CRC and adjacent normal tissues. By utilizing both bulk RNA-seq and single-cell RNA-seq data, we aimed to investigate whether the four genes displayed heightened expression levels within the tumor microenvironment. Our analysis of The Cancer Genome Atlas (TCGA) dataset, as depicted in Fig. 6A, revealed a statistically significant upregulation of these genes within CRC tissue, suggesting their pivotal involvement in the etiology and progression of CRC. Moreover, employing single-cell RNA-seq analysis, we further embarked on an exhaustive quest to elucidate the cellular heterogeneity present in 62 CRC and 36 adjacent normal colon specimens. Our meticulous efforts led to the identification of distinct cell types, including epithelial cells (n = 16,135), B cells (n = 16,134), master cells (n = 3,872), myeloid cells (n = 16,134), plasma cells (n = 16,134), stromal cells (n = 15,457), and T/NK/ILC cells (n = 16,134), as depicted in Fig. 6B. Considering that CRC lesions predominantly manifest in colorectal epithelial cells, we conducted an in-depth clustering analysis of epithelial cells in both CRC and normal contexts. Through the utilization of t-SNE visualization, we successfully demarcated the epithelial cell subcluster into two distinctive clusters (Fig. 6C). Then we focused on investigating c-MYC expression patterns within the epithelial cell subcluster, revealing notable disparities among individual cells. Specifically, upregulation of c-MYC was observed in the CRC cluster (Fig. 6D). To assess gene expression heterogeneity comprehensively, we constructed a dot plot (Fig. 6E) that captured the ratio of cells expressing specific genes to the total population while evaluating scaled mean expression levels across different labels. Quantitative analysis of c-MYC, CCND1, CDK4, and CDK6 expression in CRC and normal cells confirmed pronounced heterogeneity (Fig. 6F). Remarkably, patients were categorized into two groups based on the expression levels of these genes. Survival time, representing the duration without disease progression during and after treatment, revealed significant differences between the groups (Fig. 6G), highlighting the clinical relevance of these genes. Our bioinformatic analysis provided valuable insights into gene expression differences between CRC and adjacent normal tissues, emphasizing the therapeutic potential of targeting these four genes and the promising clinical applications of HOE.

Nowadays, the medical benefits of hemp are being increasingly recognized[23], as shown by numerous pharmacological studies that reveal its anti-tumor activities[24, 25]. However, the specific mechanism of this activity remains unclear, which poses a significant obstacle to its clinical applications.The cell cycle pathway was identified as the key biological process that was predominantly influenced by HOE, which was demonstrated to inhibit the proliferation of CRC both in vivo and in vitro at dosages with little toxicity to normal primary cells and tissues.

Notably, HOE was also found to significantly modulate the purine and pyrimidine metabolism pathway in CRC cells. Purines played a basic role in the process of the cell cycle pathway and were the main material basis of the cell cycle, which contributed to our findings from the metabolic side, further demonstrating that HOE arrested the cell cycle pathway. Previous studies have demonstrated that cannabinoids such as cannabidiol[26], THC[27], cannabigerol[26–28]which are present in the HOE, could block the progression of the cell cycle in different cell lines, providing further evidence for the reliability of our experimental results.

However, most researchers have primarily focused on the cell cycle pathway and have not explored the mechanisms in depth. In particular, our research applied the approach of a protein-protein interaction network to further delve into the hub proteins that are crucial to the cell cycle process. c-MYC was shown to be likely one of the most significant proteins. To confirm our conclusions, we examined protein levels of c-MYC in CRC cells and tumor tissues from tumor-bearing mice, and discovered HOE could reduce its level both in vitro and in vivo. Additionally, R-A. Levendal et al. reported a similar finding that cannabis could down-regulate c-MYC levels in an obese rat model [31]. c-MYC is a general transcriptional factor and oncogene, which is closely related to the development of tumors[32] and regulates some key biological processes such as cell proliferation, cell differentiation and the cell cycle[33]. Our research indicates that stabilizing c-MYC promotes G1 to S phase progression in CRC cells, which is consistent with Han et al.'s findings in human hepatocellular carcinoma cell lines.[34]. Furthermore, c-MYC modulates the transcriptional expression of CDK4, CDK6, and CCND1, which collectively regulate cellular progression through the cell cycle. Despite that c-MYC is one of the most appealing therapeutic targets for cancer treatment, its non-druggable property due to a lack of small molecule-specific active sites has limited its application[35]. our findings suggest that indirectly reducing c-MYC is a promising tactic, and they are expected to provide insight into the development of c-MYC inhibitors. The identification of principal bioactive monomer compound the from the mixture of HOE, followed by strategic chemical modifications to enhance its pharmacological properties, promisingly developed as an inhibitor of c-MYC.

Moreover, an atlas of cellular proteome and metabolome profiling was created post HOE treatment to provide an extensive profile that can serve as a significant reference point for future hemp research.. Meanwhile, the cell cycle pathway is altered after CRC cells are exposed to HOE for the reason that multiplying cells have to synthesize massive quantities of substances such as purine and pyrimidine during the cell cycle, a process that consumes an abundant amount of energy[36]. Furthermore, our findings revealed that HOE not only affects the cell cycle process, which was the primary focus of this study, but also influences other physiological processes such as oxidative phosphorylation, citric acid cycle, and pyruvate metabolism.

The investigation of natural plants as a source of ingredients that exhibit low-toxic activity and high efficiency has become a promising field of research, following the discovery and application of artemisinin and paclitaxel[37]. Our study provides a systematic approach and a fresh perspective to the pharmacological exploration of complex natural product systems, in light of the increasing discovery and investigation of biological activity components.

In summary, this study effectively elucidated the chemical composition of hemp and its therapeutic properties by utilizing UPLC-MS and GC-MS techniques to identify the primary ingredient in HOE. By utilizing proteomics and metabolomics, we were able to illustrate the changes in protein and metabolic profiles subsequent to cell exposure to HOE. Furthermore, we discovered that HOE inhibits the proliferation of colorectal cancer by blocking the cell cycle pathway via the inhibition of c-MYC, resulting in the disruption of purine and pyrimidine metabolism. Single cell results & clinical verification proved the high expression of c-MYC in the CRC tissues, emphasizing the therapeutic potential of this target and the promising clinical applications of HOE. Our results suggested that HOE could prevent cells from entering the G1 phase by reducing the levels of c-MYC protein both in vivo and in vitro. Overall, this study provides a solid theoretical and experimental foundation for the utilization of hemp in medicine.

THC

Tetrahydrocannabinol

HOE

Hemp oil extract

AIDS

Acquired immunodeficiency syndrome

CRC

Colorectal cancer

IHC

Immunohistochemistry

DEPs

Differentially expressed proteins

TMT

Tandem Mass Tags

PBS

Phosphate buffer saline

TCGA

The Cancer Genome Atlas

Ethics approval and consent to participate

The Laboratory Animal Welfare and Ethics Committee of Zhejiang University approved the protocols for animal studies (AP CODE: ZJU20220457).

Consent for publication

Not applicable.

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

The present study was supported by grants from National Natural Science Foundation of China (No. 82204584, 82304950), Zhejiang Province Human Resources and Social Security Department (Grant No.ZJ2022074).

Authors' contributions

ZS, XTF and WYJ conceived and designed this study. YHY, CY, and DJY performed the experiments and prepared the manuscript;CGX, FWL, STCY, XYD, LJ, ZY, LYJ, CY and LXS conducted the data analyses. All authors read manuscript drafts, contributed edits, and approved the final manuscript.

Acknowledgements

Not applicable.

Consent for publication

Not applicable.

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Integrated Metabolomics and Proteomics Analyses to Reveal Anticancer Mechanism of Hemp Oil Extract in Colorectal Cancer

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Abstract

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Highlights

1. Introduction

2. Materials and methods

2.1. Antibodies and reagents

2.2. Chemical profiling of HOE

2.3. Cell cultures and cell viability assay

2.4. Colony-formation assay

2.5. Animal studies

2.6. H&E staining

2.7. Immunohistochemistry (IHC) staining

2.8. TMT-labeled quantitative proteomic analysis

2.9. Untargeted metabolomics analysis

2.10. Flow cytometric analysis

2.11. Western blotting

2.12. Single-cell RNA-seq and data processing

2.13. Data statistics and analysis

3. Results

3.1. Chemical profiling of HOE

3.2. HOE inhibits CRC cell proliferation in vitro and in vivo

3.3. Quantitative proteomics-coupled with bioinformatics displays the landscape of HOE-regulated protein networks

3.4. Metabolomics reveals the profiling of HOE-regulated metabolites

3.5. HOE arrests cells in the G1 phase by inhibiting c-MYC

3.6. Bioinformatics analysis unraveled specific gene expression differences and heterogeneity in CRC

4. Discussion

5. Conclusions

List Of Abbreviations

Declarations

References

Supplementary Files

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