Research ArticleExperimental Studies

Design, Synthesis and In Vitro Anticancer Evaluation of a Stearic Acid-based Ester Conjugate

AZMAT ALI KHAN, AMER M. ALANAZI, MUMTAZ JABEEN, ARUN CHAUHAN and ALI SABIR ABDELHAMEED
Anticancer Research June 2013, 33 (6) 2517-2524;

Abstract

Aim: Chemical synthesis and characterization of a lipophilic ester conjugate, propofol stearate and evaluation of its anticancer efficacy on human breast cancer cell lines MDA-MB-361, MCF-7 and MDA-MB-231. Materials and Methods: The chemical structure of the synthesized conjugate was characterized by spectroscopic studies. Its anticancer potential was evaluated on the basis of growth inhibition, cancer cell adhesion and migration and apoptosis induction. Results: Propofol stearate exhibited significant (p<0.05) growth inhibition of breast cancer cells in a concentration-dependent manner. MDA-MB-231 cells showed highest susceptibility towards the inhibitory effect of the conjugate. Moreover, treatment of MDA-MB-231 cancer cells with 25 μM propofol stearate potentially suppressed their adhesion (~34%) and migration (~41%), and induced apoptosis (~25%). Conclusion: Exogenously-applied stearic acid as an ester derivative, inhibits the growth of human breast cancer cells and shows a beneficial role in the treatment of breast cancer, in vitro.

Anticancer drugs are frequently associated with toxicity and other side-effects (1, 2). Considering various problematic issues such as widespread systemic distribution of an administered drug and its rapid elimination, it is desirable to opt for suitable substitutes for the presently available anticancer drugs. Epidemiological reports (3-5) and animal studies (6, 7) have linked the incidence of several types of cancer to the type of fat ingested in the diet in the long term. The inhibitory effects of fatty acids on tumor development and metastasis are supported by studies using cultured cells and animal models. They demonstrate anticancer properties on various cancer cell lines including breast cancer cells (8). In vivo studies consistently support a role for dietary fat in breast cancer (9, 10). Stearic acid (C18:0) is a saturated fatty acid which is found in relatively high concentrations in several foods. It has been reported to inhibit human breast cancer cell proliferation in vitro (11-13) and breast tumorigenesis in vivo (14, 15). Stearic acid has also been shown to induce apoptosis of breast cancer cells (16) and to arrest breast cancer cell-cycle (17). Dietary stearic acid has been associated with a decrease in incidence of mammary tumor in carcinogenesis models (18). Interestingly, epidemiological studies also point to stearic acid as having potential for breast cancer prevention and treatment (19).

A combination of drugs with dietary products has attracted attention because of better responses in the treatment of cancer (20, 21). Still, drugs face a major obstacle of successful entry into the cells. To overcome this, the drug must be modified so that it can cross the plasma membrane to affect intracellular targets. Tagging of the drug to lipophilic carriers improves their efficacy by enhancing their cellular uptake and membrane transport (22, 23). Falling into the category of chemically-modified drugs, are esters of fatty acids that are lipophilic in nature. They have drawn significant attention as anticancer drug conjugates because of their lower toxicity, with specificity and potent biological activities towards various therapeutic targets (24). In previous studies, the phenolic ester conjugates of various unsaturated fatty acids were synthesized and are reported to be far more efficacious than free fatty acids against various types of cancer cell lines (25-28).

In the present study, one new fatty acid ester, namely propofol stearate, was evaluated for its ability to enhance the efficacy of cancer chemotherapy. Propofol, besides having multiple anesthetic advantages, is a potent antioxidant (29) and has been used for various biological applications (30, 31). Clinically, it reduces the metastatic probability of human cancer cells (32) and has the potential to modulate apoptosis (33). Here, the synthetic strategy followed was the same as the one used previously for the synthesis of other fatty acid esters (27, 28). Stearic acid was conjugated with propofol and the anticancer efficacy of the novel ester conjugate was elucidated on a panel of breast cancer and normal cell lines.

Materials and Methods

Chemicals. Stearic acid, 2,6-diisopropylphenol, N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, Dulbecco's modified Eagle's medium (DMEM), 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT), polyvinylidene difluoride (PVDF) membrane, sodium dodecyl sulfate (SDS), Tris, ethylene diamine tetra acetic acid (EDTA), nonidet P-40, pepstatin, ethylene glycol tetra-acetic acid (EGTA), phenyl methyl sulfonyl fluoride (PMSF), dithiothreitol (DTT) and leupeptin were purchased from Sigma-Aldrich, St. Louis, MO, USA. Cell migration and cell adhesion assay kits were obtained from Cell Biolabs, Inc., San Diego, CA, USA. Sterile filters of 0.22 μm size were purchased from Millipore, Billerica, MA, USA. Fetal calf serum was procured from Bio-Whittaker, Houston, TX, USA. Thin-layer chromatography (TLC) plates (60Å, 0.2 mm thick) and silica gel (60-120 mesh) were purchased from Fisher Scientific, Loughborough, LE, UK. Dichloromethane, n-hexane, diethyl ether, dimethyl sulfoxide (DMSO), NaCl and ethanol were procured from Merck, Frankfurter Strabe, Darmstadt, Germany. All other chemicals were of analytical grade of highest purity. Monoclonal antibodies to caspase-3 and β-actin were purchased from BD Biosciences, San Jose, CA, USA while anti-mouse peroxidase-conjugated secondary antibody was purchased from Amersham Pharmacia Biotech, UK. A chemiluminescence detection kit was purchased from GE Healthcare, Biosciences AB, Uppsala, Sweden.

Cell cultures. Breast cancer cell lines MDA-MB-361, MCF-7, MDA-MB-231 and normal breast cell line 184A1 were obtained from the American Type Culture Collection (Rockville, MD Inst). All cell lines were maintained in DMEM containing 5% fetal bovine serum and a 1% antibiotic-antimycotic solution. Cells were maintained at 37°C in a 95% humidified atmosphere containing 5% CO2. Cells were screened periodically for contamination and subcultures at 70% confluence.

Synthesis and purification of ester conjugate. The protocol of Siddiqui et al. (25) with some modifications was used to synthesize the ester conjugate. Stearic acid (1 mmol) was dissolved in 5 ml dichloromethane. The reaction mixture was stirred for 10 min at room temperature (23-25°C) after addition of 0.45 mmol of coupling reagent N,N-dicyclohexylcarbodiimide. Subsequently, 1 mmol of 2,6-diisopropylphenol (propofol) was esterified to the stearic acid reaction mixture in the presence of a catalyst, 0.152 mmol 4-dimethylaminopyridine. The reaction mixture was stirred for 10 h in the dark. Finally, the reaction was stopped by filtration and the filtrate was concentrated under reduced pressure to yield the product. Progress of reaction and synthesis of the product was visualized on TLC plates. The synthesized conjugate was purified by silica gel column chromatography with a solvent system of n-hexane and diethyl ether (1:1).

Spectral analysis. Presence of propofol in the synthesized conjugate was assessed by UV spectroscopy on a UV Mini-1240 spectrophotometer, Shimadzu, Kyoto, Japan. The absorption spectrum was measured between 200-600 nm. The infrared spectrum of the conjugate was recorded on FT-IR Nikolet-6700, Thermo Scientific, Pittsburgh, USA. For data acquisition, data spacing of 15.428 cm−1 and a resolution of 32 was used. The analysis was performed in triplicate.

MTT assay. The conjugate was examined for its cytotoxicity against three breast cancer cell lines, namely MCF-7, MDA-MB-361 and MDA-MB-231 as well as one non-cancerous cell line 184A1, using a standard MTT reduction assay (27). Cancer cells were treated with different concentrations of propofol stearate or stearic acid or propofol at a concentration range of 0-25 μM. Ethanol was used as a vehicle control and triplicate wells were prepared for each individual concentration. After treatment, the supernatant was carefully aspirated and 100 μl of DMSO was added to each well. The absorbance was measured at 620 nm and concentrations of agents that reduced 50% of cell viability (IC50) were recorded.

Cancer cell migration assay. Three concentrations (5, 15 and 25 μM) of propofol stearate were used for testing. The assay was performed with a migration kit according to the protocol of Khan et al. (28). The test conjugate and controls were supplemented with DMEM containing 10% fetal bovine serum in the lower well of the migration plate. To the inside of each insert, 100 μl of 0.5-1.0×106 cells/ml of MDA-MB-231 cell suspension was added. The plates were then incubated for 8 h at 37°C in a humidified CO2 incubator. Finally, the absorbance of 100 μl of each sample was then read at 560 nm.

Cancer cell adhesion assay. MDA-MB-231 (0.1-1.0×106 cells/ ml) cell suspension in serum-free DMEM with propofol stearate (5/15/25 μM), stearic acid, propofol or vehicle control (ethanol only) was added to the inside of each well of a pre-warmed adhesion plate. The plates were incubated for 30-90 min in a CO2 incubator and treated as per the protocol of Khan et al. (28). After air drying the wells, stained adhered cells were extracted using extraction solution. Then the absorbance of extracted sample (adhered cells) was read at 560 nm in a microtiter plate reader.

Immunoblot analysis. Cancer cells (1×107 cells per well) were grown in 6-well plates in serum-free culture medium in a humidified CO2 incubator at 37°C. After 24 h, confluent cells were treated with ethanol (control), stearic acid, propofol, 5 μM/15 μM/25 μM of propofol stearate. Following 24-h incubation, cells were harvested and washed twice in phosphate buffered saline (PBS). The cells were homogenized in 50 μl of ice-cold TNN buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.5% nonidet P-40, 1 μg/ml pepstatin, 0.5 mM EGTA, 200 μM PMSF, 0.5 mM DTT and 1 μg/ml of leupeptin). The resulting cytosolic fractions obtained after centrifugation were quantified by the BCA method (34).

Immunoblots were performed on cytosolic fractions for detecting the expression of caspase-3 as indicated. The cytosolic fractions were treated with sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS-polyacrylamide gel electrophoresis (35). The proteins were transferred to PVDF membranes by blotting and then blocked for 1 h by incubating at room temperature with PBS-T [PBS (pH 7.5) and 0.05% Tween-20] containing 5% (w/v) non-fat dry milk. The blots were incubated overnight at 4°C with the specific monoclonal antibody to caspase-3 and washed extensively with PBS-T. The blots were then incubated with anti-mouse peroxidase-conjugated secondary antibody for 2 h. The immunoreactive bands in blots were developed by enhanced chemiluminescence detection kit. Blots were re-probed with an antibody to β-actin to control for equal protein loading. The densitometric values of protein bands were quantified on Alpha Imager gel documentation system, Proteinsimple, California, USA.

Figure 1.
Figure 1.

Schematic representation of chemical synthesis of propofol stearate. A lipophilic ester conjugate was chemically designed by esterification of the terminal carboxyl group of stearic acid to the hydroxyl group of propofol. DMAP: 4-dimethylaminopyridine; DCC: N,N-dicyclohexylcarbodiimide.

Statistical analysis. Results are expressed as the means±SD in triplicates for each treatment. Individual treatments were tested against the control by using Student's t-tests. Significance was considered at p<0.05.

Results

Synthesis of an ester conjugate. The conjugate, propofol stearate, was obtained from the esterification of the hydroxyl group of propofol with the terminal carboxylic group of stearic acid. The chemical synthesis (Figure 1) resulted in a single new product. The product was observed on the TLC slide to be at a different location from that of the parent compounds. The resulting conjugate was obtained in good yield (79%) as a colorless viscous liquid (oily).

Figure 2.
Figure 2.

Ultraviolet absorbance spectra of propofol stearate. Absorbance of the parent compounds stearic acid and propofol is also shown.

Spectral characterization of propofol stearate. The UV spectra of parent stearic acid (Figure 2) had an absorption peak at 218 nm, whereas the absorption peak of propofol was observed at 280 nm. The distinct peak of the conjugate appeared at 270 nm. The fourier transform infrared (FT-IR) spectra of propofol stearate (Figure 3) had a single strong absorption peak at 1758.73 cm−1 that can be attributed to ν(C=O) and 1137.92 cm−1 that corresponds to ν(C-O) bond. The spectra also revealed a peak of aromatic C–H bond (propofol) at 2926.43 cm−1 and a short peak of aliphatic C–H bond at 2860.14 cm−1. No hydroxyl (−OH) absorption peak was seen, indicating the absence of non-esterified propofol.

Effect of propofol stearate on breast cancer cell growth inhibition. The cytotoxicity of the conjugate was tested on different types of breast cancer cells which were treated with increasing concentrations from 0-25 μM of propofol stearate. The conjugate inhibited the growth of cancer cells in a concentration-dependent manner (Figure 4). It was significant (p<0.05) cytotoxic towards breast cancer cell lines in comparison with normal 184A1 cells. The efficacy of the conjugate however varied with respect to different cell lines, where MDA-MB-231 was the most susceptible. The conjugate inhibited 50% of the cell growth (IC50) of MDA-MB-361 at 14.5 μM, MDA-MB-231 at 9.8 μM and MCF-7 at 15.8 μM.

Figure 3.
Figure 3.

The fourier transform infrared (FT-IR) spectra of propofol stearate. An aliquot of 10 μl (1 μg/μl) of conjugate was deposited exactly within the cell limit and scanned as a thin film after evaporation of the solvent.

Effect of propofol stearate on breast cancer cell migration. The results shown in Figure 5 demonstrate that stearic acid and propofol alone had minimal effect on the migration of MDA-MB-231 breast cancer cells. In contrast, propofol stearate significantly inhibited cell migration in a concentration-dependent manner by about 36% at 5 μM (p<0.05), 40% at 5 μM (p<0.05) and 41% at 25 μM (p<0.05).

Effect of propofol stearate on breast cancer cell adhesion. Cell adhesion inhibition studies were also carried out using the invasive human breast cancer cell line MDA-MB-231. Propofol stearate at the tested concentrations inhibited MDA-MB-231 cell adhesion by about 20% at 5 μM (p<0.05), 32% at 15 μM (p<0.05) and 35% at 25 μM (p<0.05) in a concentration-dependent manner. However, stearic acid and propofol alone did not significantly affect breast cancer cell adhesion (Figure 6).

Effect of propofol stearate on caspase-3 expression. Propofol stearate was able to preferentially induce expression of caspase-3 in MDA-MB-231 cells. Following treatment, a significant increase in the expression of caspase-3 (25%, p<0.05) was visualized at 25 μM concentration (Figure 7, lane 6), whereas weaker signals for caspase-3 (12% and 18%, p<0.05) were recorded at 5 and 15 μM (Figure 7, lanes 4 and 5), respectively.

Discussion

Cancer is the leading cause of death among the global population. Breast cancer is the most frequently diagnosed cancer in cancer related mortality in women. An increase in breast cancer incidence and mortality rates (36) has highlighted the need to explore alternative therapeutic strategies. Fatty acids have been known to have effects for both treatment and prevention of breast cancer in vitro and in vivo (25-28, 37). Previous studies have reported the antiproliferative effects of stearic acid, a dietary saturated fatty acid. Stearic acid inhibits proliferation of breast cancer cells and induces breast cancer cell apoptosis (11, 12, 14). In the present study, a stearic acid-based ester conjugate was synthesized by conjugating propofol to stearic acid with the aim of increasing its efficacy. Propofol was chosen because of the considerable evidence pointing to its significant benefits in combination with fatty acids in the prevention and treatment of several types of cancer in vitro and in vivo (25-28, 37). It was visualized that a conjugate of stearic acid and propofol would help in increasing efficacy towards tumors and would reduce toxicity to normal tissues.

Figure 4.
Figure 4.

Viability assessment of MDA-MB-361, MDA-MB-231, MCF-7 breast cancer cells and 184A1 normal breast cells after treatment with propofol stearate. Effects of propofol stearate along with parent stearic acid and propofol was quantified using MTT assay. Results are expressed as the mean±SD of three experiments. Significant differences from the control (p<0.05) are indicated with an asterisk.

The synthesis of the propofol stearate conjugate was straightforward where a terminal carboxylic group of stearic acid was directly coupled with the hydroxyl group present at the 1-carbon position in propofol. The presence of dicyclohexylcarbodiimide (coupling reagent) and dimethylaminopyridine (catalyst), a pre-requisite for esterification of novel conjugate, produced the conjugate in high yield. The reaction scheme is shown in Figure 1. Spectroscopic data established the chemical structure of the conjugate. The single absorption peak of propofol stearate in the ultraviolet range (Figure 2) and the presence of an ester bond (Figure 3) as visualized by FT-IR, confirmed the formation of a novel conjugate. Considering that stearic acid plays a role in tumor suppression and propofol enhances the efficacy of anticancer agents, the conjugate was tested for its anticancer effect on MDA-MB-231, MDA-MB-361 and MCF-7 breast cancer cells. The ability of the conjugate to inhibit tumor cell growth was increased compared with parental controls (Figure 4). Although the conjugate displayed potent growth inhibitory properties towards breast cancer cells, the effects on the normal human breast cell line were minimal. In order to further characterize the antiproliferative activity and likely mechanism involved, the effect of propofol stearate was investigated on MDA-MB-231 cells since they were most sensitive to the cytotoxic effect of the conjugate.

Activities such as cell adhesion and migration are essential for tumor survival. Propofol stearate was able to affect the metastatic potential of MDA-MB-231 breast cancer cells by inhibiting both migration and adhesion, whereas parental compounds alone were not effective (Figures 5 and 6). For normal cellular functioning modulation of expression of apoptotic factors must be tightly-regulated. Many anticancer therapies rely on inducing apoptosis in their target cells. Most of the caspases become activated in drug-treated tumor cells undergoing apoptosis (38). Therefore, to gain an insight whether propofol stearate mediates modulation of apoptosis, the expression of apoptotic factor caspase-3 was recorded. Caspase-3, a major executioner caspase, is used as a marker of apoptosis induction (39). The effect of propofol stearate on caspase-3 expression was especially significant, with 25% of cells expressing caspase-3 at the maximum tested concentration of 25 μM (Figure 7). The results are consistent with an already reported protective effect of stearate with respect to the risk of breast cancer (11-14). This, therefore, demonstrates that the conjugate has a marked effect on rapidly dividing cancer cells and may induce apoptosis by induction of a pathway through which it putatively modulates the aggressive behavior of breast cancer cells.

Figure 5.
Figure 5.

Effect of propofol stearate on migration of MDA-MB-231 breast cancer cells determined by cell migration assay. The extracted cells from the stained inserts were read at 560 nm in a microtiter plate reader. Results are mean±S.D. for three experiments. Significant differences from the control (p<0.05) are indicated with an asterisk.

Figure 6.
Figure 6.

Effect of propofol stearate on adhesion of MDA-MB-231 breast cancer cells determined by cell adhesion assay. The extracted sample was transferred to a 96-well microtiter plate and the absorbance was read at 560 nm. Absorbance of dye in the control (vehicle-treated) cells was regarded as 100% adherence and the percentage adherence of treated cells was calculated in comparison with that of the control cells. Results are mean±S.D. for three experiments. Significant differences from the control (p<0.05) are indicated with an asterisk.

Figure 7.
Figure 7.

A: Immunoblots showing the relative distribution of caspase-3 and β-actin (loading control) in MDA-MB-231 breast cancer cells after treatment with ethanol (control), lane 1; propofol, lane 2; stearic acid, lane 3; propofol stearate: 5 μM, lane 4; 15 μM, lane 5; 25 μM, lane 6. The protein lysate of treated cells were resolved by electrophoresis and analyzed for expression of caspase-3. To ensure equal loading, the membranes were re-probed for the presence of the housekeeping gene with β-actin antibody. B: Densitograph showing the relative density of caspase-3. Results are mean mean±S.D. in triplicates. The level of significance of differences from the control (p<0.05) are indicated with an asterisk.

Conclusion

A novel anticancer ester conjugate, propofol stearate, was designed and synthesized. The chemically-transformed stearic acid and propofol conjugate exhibited greater potency on breast carcinogenesis than did the parental compounds. The conjugate inhibited breast cancer cell growth, migration and adhesion. It also induced expression of caspase-3, an effect of apoptosis, in human breast cancer cells. The conjugate has clear antiproliferative activity, making this derivative an attractive conjugate in the treatment of breast cancer. It is also possible that this conjugate has similar effects on other cancer cell lines. Future studies need to address the effect of propofol stearate and its possible modes of action and its anticancer efficacy in animal models.

Acknowledgements

Authors would like to extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work by research grant NFG2-09-33.

  • Received March 8, 2013.
  • Revision received April 17, 2013.
  • Accepted April 22, 2013.

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Design, Synthesis and In Vitro Anticancer Evaluation of a Stearic Acid-based Ester Conjugate
AZMAT ALI KHAN, AMER M. ALANAZI, MUMTAZ JABEEN, ARUN CHAUHAN, ALI SABIR ABDELHAMEED
Anticancer Research Jun 2013, 33 (6) 2517-2524;
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