2. Materials and Methods
The transcriptional activity of genes associated with inflammatory processes in colorectal cancer cells was determined in the HT-29 cell line. The methodological part consisted of several stages. In the first stage, a culture of colorectal cancer cells (HT-29) and normal fibroblasts (NHDF) was maintained and the cytotoxicity of betulin and its derivatives (1–3) or cisplatin as a reference compound was evaluated. The lipophilicity of the compounds was also determined to assess their physicochemical parameters. The next stage of molecular analysis included the isolation of total RNA and the determination of the transcriptome in cells treated with betulin and its derivatives using oligonucleotide microarray technology.
2.1. Compound Synthesis
Betulin (purity 98%) purchased from Sigma Aldrich (Poznań, Poland) was used as a starting compound in the synthesis of derivatives
1,
2, and betulonic acid. 28-
O-Propynoylbetulin
1 was obtained by introducing a solution of DCC (
N,N’-dicyclohexylcarbodiimide, 0.23 g, 1.12 mmol) and DMAP (4-dimethylaminopyridine, 0.01 g, 0.08 mmol) in methylene chloride (1 mL) to a mixture of betulin (0.44 g, 1 mmol) and propynoic acid (0.08 g, 1.10 mmol) in a methylene chloride (CH
2Cl
2, 5 mL) (
Figure 2). The course of the reaction was monitored using thin-layer chromatography (TLC). The reaction was conducted at −10 °C for 5 h, then at room temperature for 24 h until the disappearance of betulin.
28-
O-Propargyloxycarbonylobetulin
2 was obtained by adding a solution of propargyl chloroformate (0.29 mL, 3 mmol) in chloroform (5 mL) to a mixture of betulin (0.44 g, 1 mmol) in benzene (6 mL) and pyridine (2.5 mL) (
Figure 3). The course of the reaction was monitored using thin-layer chromatography (TLC). The reaction was conducted for 4 h so that the temperature of the mixture did not rise above 5 °C. After this time, the reaction mixture was stirred at room temperature for another 24 h until the disappearance of betulin.
Crude reaction products were purified by gel column chromatography (SiO
2, chloroform/ethanol, 20:1,
v/
v, and chloroform/ethanol, 40:1,
v/
v for compounds
1 and
2, respectively). The spectroscopic data (
1H NMR and
13C NMR) (Bruker AVANCE III HD 600, Billerica, MA, USA, deuterated chloroform) and melting points (Electrothermal IA 9300, Bibby Scientific Limited, Stone, Southampton, UK) for alkynyl derivatives
1 and
2 were consistent with the previously described literature values [
4].
In the reaction of betulonic acid (0.15 g, 0.33 mmol) with 4-bromobut-1-yne (0.065 mL, 0.70 mmol) in the presence of potassium carbonate (K
2CO
3, 0.14 g, 1 mmol) in a dimethylformamide (DMF, 2.3 mL), an ester derivative of betulonic acid was obtained that was then subjected to a reduction reaction using sodium borohydride (NaBH
4, 0.40 g, 1,10 mmol) in tetrahydrofuran (THF, 10 mL) to a derivative of betulinic acid
3 (
Figure 4).
The crude reaction product was purified by gel column chromatography (SiO
2, chloroform/ethanol, 40:1,
v/
v). The spectroscopic data (
1H NMR and
13C NMR) of compound
3 were consistent with the literature information [
30]. The spectroscopic data for tested compounds
1,
2, and
3 are shown in the
Supplementary Materials (Table S1).
2.2. Cell Lines
HT-29 is a human colon adenocarcinoma cell line. They represent a valuable model due to their similarities with enterocytes of the small intestine. They have also been frequently used to study the intestinal immune response to various factors. Comparative studies on other colorectal cancer cell lines are also planned.
The culture of HT-29 ((85061109) human Caucasian colon carcinoma, Homo sapiens, human, Sigma-Aldrich, St. Louis, MO, USA) and NHDF ((CC-2511), normal human dermal fibroblasts, Homo sapiens, human, Lonza Group Ltd., Basel, Switzerland]) cells was maintained in 25 mL culture vessels with a Nunclon surface and equipped with bacterial filters (Nunc, Wiesbaden, Germany). The HT-29 cell line was cultured in McCoy’s 5A medium (Sigma Aldrich) enriched with L-glutamine and gentamicin. Additionally, the medium was enriched with 10% FBS for 24 h after cell passage. Cells from the 4th to the 6th passage were used for the experiments.
NHDF cells were cultured in FBM (Fibroblast Basal Medium; Lonza, Basel, Switzerland), enriched with FGF-B (fibroblast growth factor, Fibroblast Growth Factor-basic), L-glutamine, and gentamicin (FGM-2 TM Single Quots TM; Lonza, Basel, Switzerland). Additionally, the medium was enriched with 2% FBS for 24 h after cell passage. Cells from the 4th to the 6th passage were used for the experiments. Experiments were conducted during the logarithmic phase of cell growth and at a viability of ≥98%.
2.3. Cytotoxicity
The cytotoxicity of betulin and its alkynyl derivatives, as well as cisplatin, was measured using the In Vitro Toxicology Assay Kit, Sulforhodamine B based (TOX6, Sigma- Aldrich, St. Louis, MO, USA) according to the manufacturer’s recommendations. This test measures cell growth inhibition using the dye sulforhodamine B. Sulforhodamine B is an anionic dye that binds to the essential amino acids of cell proteins. The determination of cytotoxic activity is carried out by measuring the amount of cellular protein.
The two cell lines, HT-29 and NHDF, were passaged into 96-well plates (Nunc, Wiesbaden, Germany) at a density of approximately 6 × 103/well and incubated 24 h before the addition of the test factors. The cells were then incubated in a medium containing test compounds at a concentration of 0.1, 1, 10, or 100 μg/mL for 72 h. After 72 h, an assay was performed using the sulforhodamine B dye. Absorbance was measured at 565 nm using an MRX Revelation microplate reader (Dynex Technologies, Chantilly, VA, USA).
The derivatives of betulin and betulin were dissolved in 10% DMSO to a concentration of 1 mg/mL and next diluted in culture medium to reach the required concentrations. DMSO as a solvent did not exert any inhibitory effect on cell proliferation. The compounds in given concentrations were examined in triplicate for each experiment, which was repeated 3–5 times. The results of in vitro cytotoxic activity are expressed as an IC50 value in µg/mL. In the test, controls were untreated cells that were cultured under the same conditions. Cisplatin was used as the reference compound.
The results of the in vitro cytotoxic activity were expressed as an IC50 value (μg/mL), i.e., the concentration of a compound that inhibits the proliferation of 50% of tumor cells as compared with the control untreated cells.
2.4. Lipophilicity
RP-18F254S plates (10 × 10 cm) for lipophilicity determination were prepared by applying a starting line 1 cm from the bottom edge of the plate with a 1 cm gap from the right and left edges. The same amount of chloroform solutions of the tested substances was applied at equal distances along the starting line. The developing system used for the determinations was acetone: Tris buffer at pH 7.4. The determinations were performed in the following concentration ranges: 90%, 85%, 80%, 75%, 70%, 65%, and 60% acetone. The plates were dried under a fume hood. Then, chromatograms were visualized by spraying the plates with a solution of 10% concentrated sulfuric acid in ethanol and then heated up to 100 °C. Based on the available literature formulas, the partition coefficient (R
f), retention (R
M), and normalized retention coefficient (R
M0) values were calculated for the tested alkynyl derivatives of betulin. The experimental lipophilicity was also determined for the reference substances acetanilide, 4-bromoacetophenone, benzophenone, anthracene, and DDT. Then, based on the obtained values and the literature log P values a standard curve was made. The equations for calculating R
f, R
M, and R
M0 and received values are shown in the
Supplementary Materials (Ad.2; Table S2).
2.5. Transcriptome Determination Using Oligonucleotide Microarray Technology
Total RNA isolation from the HT-29 and NHDF cell lines was performed using TRIzol
® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. To eliminate any potential genomic DNA contamination, total RNA extracts were treated with DNase I (MBI Fermentas, Vilnius, Lithuania) on RNeasy Mini Kit columns (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quality of the extracts was evaluated by agarose gel electrophoresis using a 0.8% agarose gel stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA). The concentration and purity of nucleic acid were determined using the GeneQuant II RNA/DNA spectrophotometer (Pharmacia Biotech, Cambridge, UK). The analysis of gene expression profiles in colorectal cancer cells treated with betulin and its derivatives was performed using HG-U133A oligonucleotide microarrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Based on literature data and the Affymetrix NetAffx TM Analysis Center database (
http://www.affymetrix.com/analysis/index.affx; accessed on 31 August 2019), 973 mRNA gene IDs related to inflammatory processes that can be analyzed using HG-U133A microarray plates were selected. The criterion for considering a gene as differentiating required the absolute fold change (FC) value, which is the multiple of the fluorescence signal difference between the compared samples, to be greater than 1.1 (minimum 1.1-fold increase or decrease in signal intensity) and the significance level to be
p < 0.05.
2.6. Statistical analysis
The analysis of the cytotoxicity results used Statistica 13.1 (StatSoft).
The analysis of the results obtained using the oligonucleotide microarray technology was carried out using the MicroArray Suite 5.0 and Data Mining Tool (Affymetrix, Santa Clara, CA) programs. The normalization of the results using the robust multi-array average (RMA) method, which involves logarithmic transformation of fluorescence signal values for each transcript (log2), was performed using the RMA Express program. Statistical analysis was performed using the PL-Grid Infrastructure (
http://www.plgrid.pl/; accessed on 31 August 2019) with GeneSpring 12.0 software (Agilent Technologies UK Limited, South Queensferry, UK) and the PANTHER program (Protein ANalysis THrough Evolutionary Relationships). The samples were grouped using the hierarchical clustering method, using the measure of urban distance.
Based on the literature data and the Affymetrix NetAffx TM Analysis Center database (
http://www.affymetrix.com/analysis/index.affx; accessed on 31 August 2019), 973 mRNA IDs of genes related to inflammatory processes that can be analyzed using HG-U133A microarray plates were selected. The criterion for considering a gene as differentiating required the absolute fold change (FC) value, which is the multiple of the fluorescence signal difference between the compared samples, to be greater than 1.1 (minimum 1.1-fold increase or decrease in signal intensity) and the significance level to be
p < 0.05.
3. Results
3.1. Cytotoxicity Evaluation
The obtained cytotoxicity values are presented in
Table 1. The viability analysis results for HT-29 and NHDF cells treated with the analyzed compounds at concentrations of 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL, relative to control cells (100%), are shown in
Figures S1–S5 in the Supplementary Materials.
Betulin exhibited significant cytotoxic activity at low concentrations in the colorectal cancer cell line (HT-29). In comparison with normal fibroblast cells (NHDF), even at a concentration of 100 μg/mL betulin did not cause a 50% decrease in cell viability.
28-O-Propynoylbetulin 1 showed significant cytotoxicity against the cancer cell line HT-29 only at a concentration of 10 μg/mL, lower concentrations did not reduce cell proliferation. However, for the NHDF cell line, a concentration of 1 μg/mL of 28-O-propynoylbetulin 1 exhibited significant cytotoxicity. 28-O-Propargyloxycarbonylbetulin 2 showed the lowest cytotoxicity against NHDF cells, significantly reducing the number of cancer cells in the tested samples at concentrations of 10 μg/mL and 100 μg/mL. 3-Butynyl betulinate 3 showed noticeable cytotoxic activity only when cells were exposed to a concentration of 10 μg/mL.
3.2. Lipophilicity
Lipophilicity parameters were determined using reversed-phase thin-layer chromatography (RP-TLC). The stationary phase consisted of a nonpolar silicone oil deposited on a silica-gel-coated plate (RP-18F254S plates from Merck). The mobile phase was a mixture of acetone and an aqueous Tris buffer solution (0.2 M) at pH 7.4 in the following acetone concentration ratios: 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, and 60:40. The first step was to calculate the retardation factor (R
f). Based on the obtained R
f values, the R
M parameter values were calculated, which is a chromatographic parameter indicating lipophilicity. The relationship between R
M values and acetone concentration in the mobile phase showed a linear character with a high correlation coefficient, allowing for the determination of the R
M0 parameter. To determine the lipophilicity parameter log P
TLC in the studied system, a calibration curve was necessary. For this purpose, reference standards such as acetanilide, 4-bromoacetophenone, benzophenone, anthracene, and DDT were selected, whose log P values in the literature ranged from 1.21 to 6.38. The obtained calibration curve (
Figure 5) exhibited a high correlation coefficient, which enabled the calculation of the log P
TLC values for the tested alkynyl derivatives of betulin based on its equation.
The values of the obtained R
M0 and log P
TLC parameters for the tested compounds are summarized in
Table 2.
Figure 6 shows the correlation between the experimentally determined lipophilicity parameter log P
TLC and the biological activity of the tested compounds.
Despite the low correlation coefficient, it was observed that a lower lipophilicity may be associated with a higher biological activity for the compounds.
3.3. Evaluation of Changes in the Gene Expression Profile of Proteins Involved in Inflammatory Processes in Colorectal Cancer Cells
For the subsequent stage of the study, HT-29 cells were seeded in 6-well plates and incubated for 72 h in the presence of betulin, its derivatives, and cisplatin at a concentration corresponding to the experimentally determined IC25 value. The total RNA necessary for the microarray experiment was then extracted.
To determine the expression profile, among the 22,283 probes present on the HG- U133A microarray, those that determine the expression of genes involved in inflammatory processes (973 mRNA IDs) were selected for transcriptome analysis. The names of the corresponding probes and the information on the biological function of the genes were obtained from the Affymetrix NetAffxTM Analysis Center (
http://www.affymetrix.com/analysis/index.affx; accessed on 31 August 2019). A sample quality control was performed using a 3D PCA (3D principal component analysis) test, analysis of normalized fluorescence signal values for hybridization control probes, and 3′/5′ ratios for internal control probes. The aim of the analysis was to identify differences and similarities in the expression levels of the selected genes. The set of transcripts of genes involved in inflammatory processes, which included numerical fluorescence signal values for the modified gene expression due to exposure to the tested compounds, was analyzed to find genes with the highest FC values compared with control samples and a significance level of
p < 0.05. Comparisons were made between the following groups:
HT-29 cells treated with betulin vs. control NHDF cells;
HT-29 cells treated with derivative 1 vs. control NHDF cells;
HT-29 cells treated with derivative 2 vs. control NHDF cells;
HT-29 cells treated with derivative 3 vs. control NHDF cells;
HT-29 cells treated with cisplatin vs. control NHDF cells.
In the first stage, a cluster analysis was applied. The analyzed transcriptomes were clustered using hierarchical clustering with the Euclidean distance measure.
Clustering (
Figure 7) allowed for the comparison of changes in the expression of genes associated with inflammation in the individual analyzed groups. The group subjected to betulin treatment showed the highest similarity to the reference compound cisplatin in terms of changes in gene expression. A clear similarity in the level of gene expression was observed between the group treated with derivative
1 and the control group. The level of expression of the analyzed set of genes associated with inflammatory processes in cells exposed to derivatives
2 and
3 was different but most similar to the betulin and cisplatin groups.
The next step was to perform a sample correlation analysis (
Figure 8). It revealed the strongest correlation between changes in the gene expression profile in samples exposed to betulin and cisplatin. The largest differences were observed between the control group and the changes induced by derivative
2.
The next step in the comparative analysis of the transcriptome groups was to assess the statistical significance of the observed differences in the mRNA concentration profiles between the control groups and those exposed to betulin derivatives. The one-way analysis of variance (ANOVA) test was used to determine the set of genes that differentiated the compound-treated cells from the control cells. One-way ANOVA, with a significance threshold of
p < 0.05, identified 68 mRNA probes associated with inflammatory processes that showed significantly altered transcriptional activity compared with the control group (
Table 3).
In the subsequent statistical analysis, a post-hoc test—Tukey’s test—was performed to indicate the number of representative mRNA probes differentiating between the transcriptome groups (
Table 4).
The results obtained indicate that the largest number of differentiating mRNA probes was found in the group treated with compound 2 (18 mRNA probes). Slightly fewer probes, specifically 14 and 10 mRNA probes, were identified for the groups treated with compound
3 and cisplatin, respectively. The lowest number of differentiating mRNA probes from the control group was found for betulin. The mRNA probes identified for each group are listed in
Table 5.
The greatest expression changes (FC > 2) were observed for the HMOX1 and TMED7 mRNA IDs. In the case of cells treated with betulin, using the criteria FC > 1.1 and p < 0.05, the number of differentiating mRNA IDs was five. Among these genes, three showed increased expression and two showed reduced expression. For cells exposed to derivative 1, the number of differentiating genes was six, with four IDs showing reduced expression and two showing increased expression. Among the 18 mRNA IDs identified as differentiating the group treated with derivative 2 from the control group, 8 IDs showed increased expression, whereas the remaining 10 IDs showed decreased expression. In cells treated with derivative 3, 14 differentiating mRNA IDs were identified. Among these, seven IDs showed downregulation and seven IDs showed upregulation. In the cisplatin-treated group, out of 10 mRNA IDs, the majority (7 IDs) showed decreased expression.
The next step of the statistical analysis was to perform an overrepresentation test to determine the biological processes associated with inflammatory processes, assuming that the calculated binomial test p-value was less than 0.05.
Table 6 summarizes several biological processes involving the differentiating genes in the analyzed groups compared with the control. The most prominent differences are observed in the group of genes involved in immune cell chemotaxis, followed by genes responsible for the defense response of the immune system. The other processes show similar levels of involvement.
4. Discussion
Carcinogenesis in non-specific inflammatory bowel diseases is associated with multiple processes contributing to DNA damage, apoptosis, and the increased proliferation of cancer cells. Chronic inflammatory conditions promote the formation of a pro-tumor microenvironment that supports tumor growth, cell migration, and neoangiogenesis. Many factors, immune cells, and the cytokines and chemokines released by them are involved in this process. Inflammatory cytokines play a key role in tumor progression through multiple signaling pathways and direct effects on tumor cells, interactions with the chemokine system, promotion of epithelial–mesenchymal transition, and enhancement of metastasis. Cytokines and pro-inflammatory mediators secreted into the tumor microenvironment contribute to sustained inflammation and tumor progression by regulating transcription factors, which in turn influence the synthesis of their protein targets, thereby affecting the functions of both colorectal epithelial cells and the immune system [
31].
One of the major challenges in the successful targeted treatment of colorectal cancer is avoiding drug absorption and degradation in the upper gastrointestinal tract. Additionally, it is crucial to develop drugs that exhibit high bioavailability and low toxicity towards normal cells. In the case of colorectal cancer, a dual action of antitumor and immunomodulatory effects would be ideal, inhibiting the proliferation of cancer cells by inducing apoptosis and modulating the immune system and tumor microenvironment that promote tumor progression [
32].
Betulin is a compound that demonstrates multifunctionality. It possesses a range of biological properties, including anti-inflammatory and anticancer, as well as antioxidant, hepatoprotective, antiviral, and antiparasitic activities. New derivatives with different chemical modifications are synthesized to achieve compounds with improved biological activity. These derivatives were synthesized at the Department of Organic Chemistry of the Medical University of Silesia in Katowice. The derivatives were: 28-
O-propynoyloxybetulin
1, 28-
O-propargyloxycarbonyloxybetulin
2, and 3-butynyl betulinate. Compounds
1 and
3 were tested by Boryczka et al. for cytotoxic activity against five cell lines. The monoester derivative of betulin
2 was found to be 3–4 times more active than betulin itself on all tested cell lines (T47D—breast cancer, CCRF/CEM—lymphocytic leukemia, SW707—colorectal cancer, P388—mouse leukemia, and Balb3T3—mouse fibroblasts). Compound
1 exhibited very high cytotoxic activity against the lymphocytic leukemia cell line (IC
50 = 0.02 μg/mL) and slightly lower activity against the breast cancer cell line T47D [
4]. Compounds
1 and
2 were also tested for antitumor activity on the G-361 melanoma line. Compound
1 showed the highest activity, inducing apoptosis more than twice that of the reference betulin by activating caspase 3 [
30,
33]. An experiment evaluating the activity of compound
1 was conducted on the C32 melanoma cell line and amelanotic melanoma cell line A2058. A higher activity was observed on the A2058 line [
33]. The same derivative
1, with a propynoyl group at position C28, exhibited strong activity against human promyelocytic leukemia cell line HL-60 that was over 24 times higher than betulin [
34]. Similar results were obtained in a study involving the endometrial cancer cell line. 28-
O-Propynoylbetulin
1 showed activity similar to cisplatin. It increased NF-κB p65 expression, increased caspase 3 concentration, and inhibited collagen synthesis in endometrial cancer cells [
35].
In the first stage of the conducted research, the cytotoxicity of alkynyl derivatives of betulin was tested using sulforhodamine B dye. Among the tested compounds, betulin (IC
50 = 0.1 µg/mL) and compound
3 (IC
50 = 8.57 µg/mL) showed the most antiproliferative activity against the HT-29 cell line. The lipophilicity of the compounds was also considered, which is important for bioavailability [
36]. All compounds had a logarithm of the partition coefficient (log P) higher than five, except for betulin, which had an experimentally determined log P value below five. Therefore, the tested compounds are more suitable for local application.
The main stage of the experiment was the evaluation of changes in the expression of genes associated with inflammatory processes in HT-29 colorectal adenocarcinoma cells treated with betulin and its alkynyl derivatives 1–3.
We selected representative genes for which the significance parameter was p < 0.05 and the fold-change parameter was FC > 1.1, as a threshold to differentiate genes with altered and unaltered expression. In the HT-29 cell line, we observed an increase in fluorescence signal for three mRNA IDs and a decrease in the fluorescence signal for two mRNA IDs concerning betulin. We identified differential expression of six mRNA IDs compared with control cells when HT-29 cells were treated with compound 1. We observed a significant decrease in the fluorescence signal for the ICAM1 mRNA ID, signifying a reduction in the expression of this gene. In the group of cells treated with compound 2, the expression of 18 mRNA IDs altered significantly in the studied cell line. Compound 3 caused alterations in the expression of 13 mRNA IDs. The final group comprised cells that were exposed to cisplatin, which was used as a reference compound. Its activity caused changes in the expression of 10 mRNA IDs.
In the groups treated with derivatives
2 and
3, betulin, and cisplatin, a single mutual CHUK mRNA ID was selected, demonstrating increased expression in all groups in comparison with the control. For the group treated with substance
1, two mRNA IDs were exclusive to this group: HMOX1 and CXCR4. The expression of HMOX1 mRNA is stimulated by a variety of molecular and physical signals. The significance of HMOX1 in the pathogenesis of colorectal cancer remains unclear. These pathways are activated by the biologically active catabolites of heme degradation and promote disease progression through anti-apoptotic, antioxidant, and anti-inflammatory effects in addition to proangiogenic and prometastatic effects. There are instances in the literature where the protein encoded by HMOX plays an opposing anti-tumoral role. However, it is assumed that HMOX1 acts as a conditional tumor promoter in the pathogenesis of colorectal cancer [
37,
38].
The C-X-C chemokine receptor 4 (CXCR4) has been suggested to play an important role in several types of cancer and has been implicated in biological behaviors associated with tumor progression. In human pancreatic ductal adenocarcinoma and colorectal cancer, CXCR4 signaling can suppress the immune system and impair the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells [
39,
40].
Three ID mRNAs were selected as specific for cells incubated with betulin (CD59, DARC, and CEBPB) and one ID mRNA (IL25) was downregulated by both betulin and compound
2. CD59 encodes a glycoprotein on the cell surface that regulates cell lysis mediated via the complement system and is involved in lymphocyte signal transduction. CD59 additionally regulates the function, infiltration, and phenotypes of various immune cells in the tumor microenvironment. Increased CD59 expression on malignant colonic cells may protect against complement-mediated damage, thereby enabling higher local invasiveness and metastasizing potential. An increased level is a disadvantageous treatment factor [
41,
42].
The role of DARC in accelerated inflammation and malignant transformation remains understudied. Multiple studies show that the expression of DARC is downregulated in colorectal cancer and is associated with the clinical and pathological characteristics of this disease [
43,
44].
IL-25 might play a multifarious role in cancer progression or regression. Although IL-25 inhibits tumor growth in some cases, it has been shown in several studies that IL-25 also plays a crucial role in cancer progression. The tumor-suppressive role of this interleukin is mainly correlated with the infiltration of eosinophils and B cells into the tumor microenvironment and induction of apoptosis. In contrast, its tumor-supportive roles rely on the deviation of immune responses and cell growth [
45].
Ten ID mRNAs are specific only for a group of genes associated with inflammatory processes after the action of compound 2, which differentiates them from the others. Reduced expression of the ICAM1 gene was observed for three of the study groups: 3 vs. control, 2 vs. control, and 1 vs. control. Intracellular adhesion molecule-1 (ICAM-1) is a potential therapeutic target to enhance therapeutic effectiveness for colorectal cancer. Recent studies have shown that it promotes malignancy in several carcinomas. ICAM-1 may further accelerate SRC signaling, promoting the malignant potential of cancer, so it can be considered as a novel therapeutic anticancer target [
46].
In the group with the third derivative vs. control, seven mRNA IDs were isolated specific to this group.
In the overrepresentation analysis, nine mRNA IDs related to oxidative stress response, antimicrobial response, immune defense, chemotaxis, and cytokine signaling pathway were selected. Among them was the CHUK gene encoding the NF-κB kinase inhibitor. The role of the NF-κB pathway has been confirmed in the development of many cancers. It is activated by various stimuli, including pro-inflammatory cytokines, bacterial and viral products, DNA damage, and oxidative stress. It activates the transcription of hundreds of genes involved in immune response, growth control, and protection against apoptosis. It is also responsible for chemotherapy and radiotherapy resistance. The increased expression of the NF-κB inhibitor observed under the action of betulin and compounds
2 and
3 will prevent the activation of NF-κB molecules and their translocation into the cell nucleus. This is a confirmed direction of the actions in colorectal cancer therapy [
47]. Another gene highlighted in the overrepresentation analysis was MAP2K3. It encodes the protein kinase mitogen-activated protein kinase 3, belonging to the MAP kinase family. Activation of MAP kinases leads to cell proliferation and survival. Many studies emphasize the role of MAP2K3 in tumor progression and invasiveness, indicating it as a promising target for anticancer therapy. Silencing this gene reduces the viability of cancer cells without affecting normal cells. Moreover, it reduces tumor growth and improves the biological response to chemotherapy agents [
48]. In the presented study, in the microarray experiment, a decrease in mRNA expression of MAP2K3 was observed in HT-29 cells subjected to the action of compound
2 and cisplatin.
The analysis of individual changes does not allow us to clearly determine the effect of individual derivatives on colorectal adenocarcinoma cells of the HT-29 cell line. However, it shows the differences in the changes that occur under the influence of individual alterations in the chemical structure.