Paclitaxel, a chemotherapeutic agent used in the treatment of breast cancer and other solid tumor types, including ovarian and lung, causes a dose-dependent neuropathic pain, which limits its use. Chemically modified tetracycline-3 (COL-3) has anticancer properties and was previously reported to inhibit neuroinflammation and protect against paclitaxel-induced neuropathic pain (PINP) in mice models. However, it is not known whether it affects the anticancer activities of paclitaxel. Thus, the aim of the present study was to evaluate the effect of COL-3 on the anticancer activity of paclitaxel on the breast cancer cell lines MCF-7 (estrogen receptor-positive), pII [estrogen receptor-negative (ER-ve)] and MDA-MB-231 (ER-ve). Cell proliferation, apoptosis and cell cycle stage were determined using an MTT assay, Annexin V/7-aminoactinomycin D and flow cytometry. The expression of various signaling molecules was determined with ELISA-based proteome profiling and western blotting. Additionally, the degree of cell invasion was determined with a Matrigel assay and caspase-3 activity was determined with a colorimetric assay. Treatment with paclitaxel or COL-3 alone inhibited cell proliferation in a concentration-dependent manner in all cell lines. The anti-proliferative effects of paclitaxel and COL-3 in combination varied from synergism against MDA-MB-231 and pII cells to notably additive and slight antagonism against MCF-7 cells. In the highly proliferative and invasive pII cells, the observed synergistic anti-proliferative effect was partially through the induction of apoptosis via modulation of caspase-3 levels and activity, and P70S6K phosphorylation, but not cell cycle arrest. COL-3 inhibited the invasion of pII cells in a concentration-dependent manner partially through inhibiting total matrix metalloproteinase activity. The combination regimen significantly inhibited the expression of two proteases, ADAM metallopeptidase with thrombospondin type 1 motif 1 and proteinase 3. In conclusion, the combination of paclitaxel and COL-3 indicated additive to synergistic anti-proliferative effects on breast cancer cells mediated partially via the induction of apoptosis. The combination regimen could further inhibit invasion and metastasis. Thus, COL-3 could be a beneficial adjunct to a paclitaxel-based anticancer regimen to improve therapeutic outcome and reduce the adverse effects of paclitaxel, primarily PINP.
Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer mortalities among females based on the estimates of cancer incidence and mortality globally for 36 cancer types in 185 countries, which was produced by the International Agency for Research on Cancer in 2018 (
Chemotherapeutic regimens primarily consist of a combination of ≥2 cytotoxic drugs (
The use of paclitaxel is associated with a number of serious dose-dependent side effects, including paclitaxel-induced peripheral neuropathy (PIPN), which may necessitate dose reduction or withdrawal during the course of chemotherapy (
The human breast carcinoma cell lines MCF-7 (ER+ve) and MDA-MB-231 (ER-ve;
COL-3 (purchased from Galderma, Research and Development SNC, Les Templier, France) was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 1 mM and stored at −20°C in aliquots. Paclitaxel, purchased from Tocris Bioscience (Bristol, UK), was dissolved in DMSO to a stock concentration of 1 mM and stored at −20°C in aliquots. All experimental incubation with drugs was conducted at 37°C, 5% CO2 and 95% humidity in an incubator. The control vehicle used was 0.01% DMSO.
The effect of various concentrations of paclitaxel (1, 2.5, 5, 10, 25, 50, 100, and 1,000 nM), COL-3 (50, 100, 1,000, 2,500, 5,000, 10,000, and 20,000 nM) or their combination on cell proliferation was examined using a colorimetric MTT assay (Promega Corporation, Madison, WI, USA), as previously described (
The effect of COL-3, paclitaxel or their combination on pII cell apoptosis was measured using Annexin V/7-aminoactinomycin D apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA), as previously described (
Subsequently, ~1×104 pII cells were seeded in triplicate wells and incubated overnight at 37°C in an atmosphere containing 5% CO2 and 95% humidity. The cells were then treated with the vehicle or various concentrations of paclitaxel, COL-3 or their combination. After 72 h of incubation at 37°C in an atmosphere containing 5% CO2, cells were trypsinized and washed once with ice-cold PBS. The pellet was then re-suspended with PBS and fixed by adding ice-cold 70% ethanol while vortexing at 14,000 × g for 20 sec at 4°C. The samples were then stored at −20°C overnight. The following day, samples were centrifuged at 66 × g for 15 min at room temperature and washed once with PBS. Pellets were treated with RNase, incubated for 15 min at 37°C and 200 µl propidium iodide solution (DNA Prep stain kit; Beckman Coulter, Inc., Brea, CA, USA) was added. The samples were analyzed using a Cytomics FC500 flow cytometer with a maximum emission of 605 nm. The DNA content of cell duplicates during the S phase of the cell cycle, and the relative amount of cells in the G0 and G1 phases, in the S phase, and in the G2 and M phases were determined utilizing the fluorescence of cells in the G2/M phase, which were twice as high as that of cells in the G0/G1 phase. This was obtained using CXP Software, version 2 (Beckman Coulter, Inc.).
The degree of pII cell invasion through the basement membrane matrix was determined using the Cultrex® 24-Well basement membrane extract (BME) Cell Invasion assay kit (Trevigen, Haithersburg, MD, USA). All procedures and reagent preparations were conducted according to the manufacturer's protocols. Briefly, insert membranes were coated with 1X BME and incubated at 37°C overnight. pII cells, that had been serum-starved overnight, were re-suspended at 1×106 cells/ml in Advanced DMEM with or without drugs (100, 1,000, 5,000 and 10,000 nM), and 100 µl (1×105 cells) was loaded into the upper chambers. To the bottom chamber, 500 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS was added as a chemoattractant. After 24 h of incubation at 37°C, the media in the top chambers as well as the bottom chambers were aspirated followed by washing with 1X wash buffer. Subsequently, 500 µl Cell Dissociation Solution/Calcein-acetomethylester (AM) complex was added to the bottom chamber of each well and incubated at 37°C in an atmosphere containing CO2 for 60 min. Cells internalize Calcein-AM and intracellular esterases cleave AM moiety generating free calcein, which fluoresces. The degree of cell invasion was determined by recording the fluorescence emission using a microplate reader (Luminometer) with a filter set of excitation/emission at 485/535 nm.
A total of three different proteome profiler array kits, Human protease array kit, Human phospho-kinase array kit and Human apoptosis array kit (R&D Systems, Inc., Minneapolis, MN, USA), were used to determine the expression levels of a number of groups of proteins in cell extracts/lysates following the manufacturer's protocols.
Subsequently, ~1×106 pII cells/well were cultured for 24 h at 37°C in an atmosphere containing 5% CO2 under the following treatment conditions: Vehicle (control); 2.5 nM paclitaxel; 5 µM COL-3; or 2.5 nM paclitaxel and 5 µM COL-3. Upon removal of the advanced DMEM, the cell monolayers were washed once with ice-cold PBS and then lysed using lysis buffer supplemented with protease inhibitors [1 µg/ml leupeptin, 1 µg/ml aprotinin and 10 µg/ml phenylmethylsulfonyl fluoride (PMSF); Sigma-Aldrich; Merck KGaA, Darmstadt, Germany]. Cells were harvested using a sterile disposable rubber cell scraper and transferred into Eppendorf® tubes. The cell lysates were then centrifuged at 14,000 × g for 10 min at 4°C, and the supernatant was transferred to new Eppendorf tubes for subsequent protein analysis or stored at −80°C for later analysis. Total protein concentration in the cell lysates was determined with a standard Bradford assay.
The relative phosphorylated/expression levels of 45 kinases (cat. no. ARY003B), 35 apoptosis-associated proteins (cat. no. ARY009) and 35 proteases (cat. no. ARY021B) (R&D Systems, Inc.) were detected using Proteome Profiler™ Human Protease Array kits aforementioned, according to the manufacturer's protocols.
The general activity of MMPs was determined using the MMP activity assay kit from Abcam (cat. no. ab112146; Cambridge, UK), as previously described (
Cells were seeded (0.1×106 cells) in 6-well plates to 80% confluence, and then incubated with 2.5 nM paclitaxel, 5 µM COL-3 or a combination regimen (2.5 nM paclitaxel + 5 µM COL-3) for 48 h at 37°C in an atmosphere containing 5% CO2. Cells were then trypsinized, pelleted at 66 × g for 15 min at room temperature and DNA was isolated from the cells using a DNA isolation kit (Qiagen, Inc., Gaithersburg, MD, USA), according to the manufacture's protocol. Subsequently, 1 µg DNA was run on 2% agarose gel stained with ethidium bromide at 50 V, and the gel was examined under an UV trans-illuminator. Additionally, a 1 µg DNA ladder was also included in the experiment (λ DNA/
Cells were cultured (0.1×106 cells; at 37°C in an atmosphere containing 5% CO2) in 6-well plates to an 80–90% confluence, and treated with vehicle (control), 2.5 nM paclitaxel, 5 µM COL-3 or combination regimen poly(ADP-ribose) polymerase (2.5 nM paclitaxel + 5 µM COL-3) for 48 h. The medium was subsequently aspirated off, and cell monolayers were harvested by scraping and re-suspension into 300 µl lysis buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X, 100 µg/ml PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin, and then stored at −80°C. Protein concentration was determined with a Bradford assay using bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) as the standard, and 3 µg protein lysate was mixed with an equal volume of 2X SDS and incubated at 90°C for 10 min. Samples were then loaded onto 10% SDS-PAGE and electrophoresed at 150 V for 1 h. Subsequently, proteins were transferred to a nitrocellulose membrane and blocked with 2% BSA for 1 h at room temperature prior to being incubated overnight at 4°C with primary antibodies (prepared in 2% BSA) against β-actin (loading control; 1:1,000 dilution), or phospho- or total- B-cell lymphoma-2 (BCL-2)-associated X (BAX), BCL-2 associated agonist of cell death (BAD), BCL-2 antagonist/killer (BAK), BCL-2, cytochrome
Cells (1×106) were treated with paclitaxel (2.5 nM), COL-3 (5 µM) or a combination regimen (2.5 nM paclitaxel + 5 µM COL-3) for 48 h at 37°C in an atmosphere containing 5% CO2 and then collected by trypsinization and washed twice with ice-cold PBS. Subsequently, the cells were centrifuged at 42 × g for 5 min at 4°C and the pellet was washed with ice-cold lysis buffer. The protein concentration was calculated using a Bradford assay. Caspase-3 activity in the lysate of the different treatment conditions was determined using a caspase-3 colorimetric assay kit from GenScript (cat. no. L00289; Piscataway, NJ, USA), and was expressed as caspase-3 activity/mg of protein.
Statistical analyses were performed using one-way analysis of variance followed by Dunnett's (for comparing between treatment conditions) or Bonferroni's (for comparing within treatment conditions) Multiple Comparison Test using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. Using the GraphPad Prism software, the concentration of paclitaxel or COL-3 that produced the half-maximal response (IC50) was calculated using non-linear regression analysis. The data were fitted to a dose-response-inhibition equation [log (inhibitor) vs. normalized response curve]. Results were expressed as the mean ± standard error of the mean.
To test for synergism, summation or antagonism, the combination index (CI) was calculated by adapting the Chou and Talalay equation for mutually non-exclusive drugs: CI = (D)1(Dx)1+(D)2(Dx)2+(D)1 (D)2(Dx)1 (Dx)2, where CI<1 indicates synergism, CI=1 indicates summation and CI>1 indicates antagonism (
Paclitaxel inhibited cell proliferation in all of the tested cell lines in a concentration-dependent manner (
COL-3 inhibited cell proliferation in all of the tested cell lines in a concentration-dependent manner (
The effects of various combination regimens on MCF-7 cell proliferation varied from notably additive to a slight antagonism. Combining 2.5 nM paclitaxel with 5 µM COL-3 resulted in a notably additive effect (CI=0.99) although the combination regimen did not significantly inhibit proliferation, compared with treatment with COL-3 alone (62 vs. 58%, respectively; P>0.05;
For pII cells, the combination regimen resulted in synergistic inhibitory effects on cell proliferation. Combination of 2.5 nM paclitaxel with 5 µM COL-3 resulted in a synergistic effect (CI=0.51;
With regards to MDA-MB-231 cells, the combination regimen also resulted in synergistic inhibitory effects on cell proliferation. Synergism was achieved when combining 2.5 nM paclitaxel with 1 µM COL-3 (CI=0.45;
Notably, the ER-ve cell lines were more responsive to the combination regimens, which resulted in synergistic inhibitory effects on cell proliferation, compared with the ER+ve cell line.
The rational for using different concentrations of COL-3 or paclitaxel was due to the different sensitivities of the cell lines to the effects of the drugs. A concentration, which inhibited cell proliferation by ~50% in the combination regiments, was selected for the subsequent experiments.
At the tested concentrations, treatment with 2.5 nM paclitaxel or 5 µM COL-3 alone significantly decreased viable cells and increased early/late apoptotic cells, compared with treatment with vehicle (P<0.05;
Treatment with paclitaxel at a concentration of 100 nM, significantly decreased (P<0.01) the cells in the G0/G1 phase, from 75.3±1.2% to 8.4±1.9%, and increased (P<0.05) cells in the G2/M phase, from 12.0±0.5% to 27.8±1.1%, compared with the vehicle treatment; thus, it arrested the cell cycle at the G2/M phase (
No statistically significant effects on the expression levels of phosphokinases or apoptosis-associated proteins were observed following treatment with paclitaxel, COL-3 or their combination, which may be due to variation in the expression of phosphokinases or proteins between the samples analyzed (
Among the 35 tested proteases, the expression of the proteases ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1) and proteinase 3 (PR3) was significantly reduced by the combination regimen, compared with the vehicle-treated cells (61 and 48% respectively; P<0.05;
Treatment with paclitaxel, but not COL-3, significantly increased caspase-3 activity in pII cells, compared with vehicle treatment (P<0.05). The combination regimen resulted in an enhanced effect and significantly increased caspase-3 activity, compared with monotherapy (P<0.05;
EGF stimulation of pII cells significantly increased the total MMP activity, compared with vehicle treatment (P<0.05;
COL-3 produced a concentration-dependent inhibitory effect on pII cell invasion in the Cultrex® 24-well invasion assay and reached statistical significance at 10 µM, compared with vehicle (78% inhibition; P<0.01;
The present study demonstrated that the chemically modified tetracycline COL-3 enhances the anticancer effects of paclitaxel against ER+ve and ER-ve breast cancer cell lines. COL-3 on its own had anti-proliferative effects against ER+ve and ER-ve breast cancer cell lines, although it was less potent than paclitaxel. There was synergistic anti-proliferative activity between COL-3 and paclitaxel against ER-ve breast cancer cell lines, and the anti-proliferative effects varied from notably additive to slight antagonism against the ER+ve cell line. Reduced concentrations of the drugs in combination produced similar anti-proliferative activity, compared with high concentrations of paclitaxel or COL-3 alone. The synergistic anti-proliferative activity of the drug combination against the highly invasive and proliferative ER-ve breast cancer cell line pII may be due to the enhanced anti-apoptotic activity, but not cell cycle arrest. Additionally, COL-3 inhibited pII cell invasion and MMP activity
Although paclitaxel is considered an integral agent in the treatment of breast cancer and other solid tumor types, the dose-dependent side effects associated with the development of peripheral neuropathy (PIPN) limits its use and may render the treatment ineffective (
Paclitaxel significantly inhibited cell proliferation in all the tested cell lines in a concentration-dependent manner, which is consistent with previous
The combination regimens of paclitaxel and COL-3 had synergistic anti-proliferative effects against ER-ve cell lines and the anti-proliferative effects varied from notably additive to slightly antagonism on the ER+ve cell line. The synergistic anti-proliferative effect observed against the ER-ve pII cells was partially via the induction of apoptosis rather than inducing cell cycle arrest. Additionally, it was possible to reduce the concentrations of paclitaxel as well as COL-3 when the drugs were used in combination, while producing the identical degree of inhibition in cell proliferation as increased concentrations of each drug alone. Thus, the combination could assist in reducing the risk of developing dose-dependent side effects, including PIPN. It may also be possible to reduce the risk of developing cutaneous photo-toxicity, which is the dose-limiting side effect of COL-3 (
Numerous studies demonstrated that paclitaxel kills cancer cells through the induction of apoptosis (
It is considered that stabilization of microtubules of paclitaxel induces mitotic arrest at the G2/M phase of the cell cycle (
COL-3 treatment significantly reduced cell invasion partially through inhibiting total MMP activity. CMTs have been demonstrated to inhibit the enzymatic activities of gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10 and MMP-11), collagenases (MMP-1, MMP-8 and MMP-13) as well as non-collagenolytic proteases (
In conclusion, the present study indicates that COL-3 potentiates the anticancer activity of paclitaxel by enhancing its inhibitory effects on cell proliferation, inducing apoptosis as well as inhibiting invasiveness. The molecular mechanism may involve the modulation of the expression of proteases, including ADAMTS1 and PR3, caspase-3 expression/activity and P70S6K phosphorylation. Furthermore, the combination regimen would also offer opportunities for the reduction in the effective dose of paclitaxel, which in turn would have further beneficial effects for the reduction of the dose-dependent side effects, including PIPN. This indicates that the combination of paclitaxel and COL-3 in addition to reducing the development of PIPN, as recently reported (
The authors would like to acknowledge the Core facility in the Health Science Center (Kuwait University, Safat, Kuwait) for the technical assistance.
This work was supported by grants from Kuwait University Research Sector (grant nos. YP01/14 and SRUL02/13).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MAK and WM conceived and designed the experiments. RMET, PM and MAK performed the experiments. RMET, MAK, PM and WM analyzed the data. WM and MAK contributed reagents, materials and analysis tools. RMET, MAK and WM wrote the paper. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of monotherapy with paclitaxel or COL-3 on cell viability. Growth was assessed with an MTT assay after 72 h of incubation with drugs or their vehicle. (A) MCF-7 cell viability following treatment with vehicle and different concentrations of paclitaxel. Each bar represents the mean ± standard error of the mean of 3–8 determinations. (B) pII cell viability following treatment with vehicle and different concentrations of paclitaxel. Each bar represents the mean ± standard error of the mean of 3–8 determinations. (C) MDA-MB-231 cell viability following treatment with vehicle and different concentrations of paclitaxel. Each bar represents the mean ± standard error of the mean of 12 determinations. (D) MCF-7 cell viability following treatment with vehicle and different concentrations of COL-3. Each bar represents the mean ± standard error of the mean of 6–10 determinations. (E) pII cell viability following treatment with vehicle and different concentrations of COL-3. Each bar represents the mean ± standard error of the mean of 4–13 determinations. (F) MDA-MB-231 cell viability following treatment with vehicle and different concentrations of COL-3. Each bar represents the mean ± standard error of the mean of 9 determinations. *P<0.05 and **P<0.01, compared with the vehicle-treated group. COL-3, chemically modified tetracycline-3.
Effect of combination regiments on cell viability. Growth was assessed with an MTT assay after 72 h of incubation with drugs or their vehicle. (A) MCF-7 cell viability following treatment with vehicle and different concentrations of paclitaxel and COL-3 alone and in combination. Each bar represents the mean ± standard error of the mean of 12–20 independent determinations. (B) pII cell viability following treatment with vehicle and different concentrations of paclitaxel and COL-3 alone and in combination. Each bar represents the mean ± standard error of the mean of 9–12 independent determinations. (C) MDA-MB-231 cell viability following treatment with vehicle and different concentrations of paclitaxel and COL-3 alone and in combination. Each bar represents the mean ± standard error of the mean of 9–21 independent determinations. **P<0.01, compared with the vehicle-treated group. #P<0.05, ##P<0.01, compared with the monotherapy groups. COL-3, chemically modified tetracycline-3; pac, paclitaxel; ns, not significant.
Effect of combination regimens on pII cell apoptosis. Apoptosis was determined using PE Annexin V/7-AAD. Samples were analyzed by flow cytometry after 72 h of incubation with the vehicle, 2.5 nM paclitaxel, 5 µM COL-3 or their combination. (A) Cell viability following treatment with vehicle or drugs. (B) Early apoptotic cells following treatment with vehicle or drugs. (C) Late apoptotic cells following treatment with vehicle or drugs. Each bar represents the mean ± standard error of the mean of 6 independent determinations. (D) Quadrant graphs of the Annexin V/7-AAD data depicted in panels A-C. (E) DNA fragmentation image for the different treatment groups along with the densitometric analysis (using ImageJ software). Using a DNA ladder, it was observed that the DNA fragments produced by the combination regimen are >23,130 bp. Each bar represents the mean ± standard error of the mean of 3 independent determinations. *P<0.05, compared with the vehicle-treated group. #P<0.05, compared with combination and individual drugs. COL-3, chemically modified tetracycline-3; pac, paclitaxel; 7-AAD, 7-aminoactinomycin D.
Effect of combination regimens on pII cell cycle. Cells were seeded in a 24-well plate, allowed to attach overnight and treated after 24 h with vehicle or different concentrations of (A) paclitaxel (2.5 and 100 nM), (B) COL-3 (5 and 10 µM) or (C) their combination (2.5 nM paclitaxel and 5 µM COL-3). Samples were then analyzed by flow cytometer after 72 h of incubation with the drugs. Each bar represents the mean ± standard error of the mean of 6 independent determinations. *P<0.05 and **P<0.01, compared with the vehicle-treated group. (D) Flow cytometry images for the various treatment groups. COL-3, chemically modified tetracycline-3.
Effect of combination regimens on the phosphorylation levels of tested kinases and apoptosis-associated proteins in pII cells. Lysates of cells were collected after 24 h of incubation with the tested drugs. Densitometric values are means normalized to the mean of the reference spots on each blot, and the percentage of relative expression levels of selected phosphokinases and apoptosis-associated proteins was determined (vehicle set as 100%) (A) Expression levels of p70S6 kinase (T389), eNOS, EGFR and HSP27. (B) Expression levels of p53 at S392 and S46. (C) Expression levels of ADAM8, ADAMTS1, ADAMTS13, protein-convertase 9 and proteinase 3. (D) Expression levels of cathepsins E, L and S, and kallikreins 6 and 7. (E) Expression levels of MMPs −3, −8, −9 and −10. Each bar represents the mean ± standard error of the mean of 4 independent experiments. *P<0.05, compared with the vehicle treated group. eNOS, epithelial nitric oxide synthase; EGFR, epidermal growth factor receptor; HSP27, heat shock protein 27; COL-3, chemically modified tetracycline-3; MMP, matrix metalloproteinase; ADAMTS1, ADAM metallopeptidase with thrombospondin type 1 motif 1.
Effect of drug treatment on the expression and phosphorylated levels of various signaling molecules and caspase-3 activity. (A) Cells were treated with paclitaxel, COL-3 or combination regimen for 48 h. Total protein lysate (3 µg) was electrophoresed on 10% SDS-PAGE, blotted onto nitrocellulose membrane and probed with antisera to P- or T-BAX, BAD, BAK, BCL-2, cytochrome
Effect of drug treatment in total MMP activity and invasion of pII cells. (A) Cells were left untreated (UT) or stimulated with EGF (100 ng/ml) with or without pre-treatment with individual drugs or combination regiment, and total MMP activity was determined in the supernatant. Histobars represent the mean ± SEM of 6 independent determinations, *P<0.05, compared with the UT group. #P<0.05, compared with the EGF/vehicle treated group. (B) Cells were left untreated (vehicle) or exposed to various concentrations of COL-3. The total number of pII cells penetrating through a basement membrane extract towards serum components was determined by fluorometric analysis. Histobars represent the mean ± SEM of 6 independent determinations. **P<0.01, compared with the vehicle-treated group. SEM, standard error of the mean; COL-3, chemically modified tetracycline-3; pac, paclitaxel; MMP, matrix metalloproteinase; UT, untreated; EGF, epidermal growth factor.
Comparison of the anti-proliferative effects of combined concentrations of pac and COL-3 with the calculated individual concentrations of pac or COL-3 that would produce equivalent effect as combination for all the cell lines.
Calculated individual concentration that produces equivalent % inhibition of cell viability as the combination regimen | Folds of reduction of the drug concentration in the combination regimen vs. individual drug treatment | ||||||
---|---|---|---|---|---|---|---|
Cell lines | Combined concentrations | % inhibition of cell viability of the combination regimen | Pac (nM) | COL-3 (µM) | CI | Pac (nM) | COL-3 (µM) |
MCF-7 | 2.5 nM pac + 5 µM COL-3 | 62.1 | 58.8 | 6.6 | 0.99 | 23.5 | 1.32 |
5 nM pac + 2.5 µM COL-3 | 43 | 27.2 | 3 | 1.17 | 5.44 | 1.2 | |
10 nM pac + 2.5 µM COL-3 | 49 | 34.6 | 4 | 1.09 | 3.5 | 1.6 | |
pII | 2.5 nM pac + 5 µM COL-3 | 63.1 | 14.7 | 17.2 | 0.51 | 6 | 3.4 |
5 nM pac + 5 µM COL-3 | 67.8 | 18.1 | 21.3 | 0.58 | 4 | 4.3 | |
10 nM pac + 5 µM COL-3 | 71 | 21.2 | 24.9 | 0.767 | 2.1 | 4.98 | |
MDA-MB-231 | 2.5 nM pac + 1 µM COL-3 | 59.5 | 9.6 | 6.5 | 0.45 | 3.8 | 6.5 |
2.5 nM pac + 2.5 µM COL-3 | 56.7 | 8.5 | 5.8 | 0.85 | 3.4 | 2.3 |
CI, combination index; COL-3, chemically modified tetracycline-3; pac, paclitaxel.