ORIGINAL ARTICLE Year : 2021  |  Volume : 46  |  Issue : 3  |  Page : 185-191

In vitro comparison of the cytotoxic effects of lenalidomide alone and in combination with verapamil on myeloma cell line

Seyma Tastemur MD 1, Mehmet Şencan2, Hatice Terzi2, Merve Ergül3, Mercan Taştemur4
1 Department of Internal Medicine, Sivas Numune Hospital, Ankara City Hospital, Ankara, Turkey
2 Department of Internal Medicine, Discipline of Hematology, Faculty of Medicine, Ankara City Hospital, Ankara, Turkey
3 Department of Pharmacology, Faculty of Pharmacy, Sivas Cumhuriyet University, Sivas, Turkey
4 Department of Internal Medicine, Discipline of Geriatrics, Ankara City Hospital, Ankara, Turkey

Date of Submission22-Jan-2021Date of Acceptance26-Jan-2021Date of Web Publication13-May-2022

Correspondence Address:
Seyma Tastemur
Yesşilyurt mahallesi SŞifa caddesi, Sivas Numune Hospital, Sivas, Merkez
Turkey

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/ejh.ejh_4_21

Introduction Multiple myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells. High-dose chemotherapy with novel agents and autologous stem cell transplantation are options for treatment. However, MM treatment generally results in failure. The most important reason for this failure is the resistance to chemotherapeutic drugs. Various studies have been tried to combine chemosensitizer agents that increase the cytotoxic effects of the chemotherapeutics to eliminate the drug resistance. In our study, we aimed to evaluate the effect of verapamil on the cytotoxic effect of lenalidomide on the myeloma cell line.
Materials and methods Verapamil is a chemosensitizer that suppresses the P-glycoprotein. In our study, lenalidomide, an immunomodulatory agent, was compared alone and in combination with verapamil for cytotoxic effects. U266 MM cell line was used in the study. At the concentrations of 0.001, 0.01, 0.1, 1, 10, 50, and 100 µM, lenalidomide alone and the combination of lenalidomide at the same concentrations with 2.5 µg/ml of verapamil were compared in terms of possible cytotoxic properties. Cell viability was measured by XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test.
Results A statistically significant decrease in the inhibitor concentration, causing 50% decrease in cell proliferation (IC50) of lenalidomide, was provided via verapamil administration. Our study revealed that the cytotoxic effect of lenalidomide increases when combined with verapamil.
Conclusion We aimed to understand whether the cytotoxic effect of lenalidomide, which has an important place in the treatment of MM, can be increased with an easily available drug such as verapamil. We think that more studies and meta-analyses are needed owing to the different results related to the subject in the literature, and we hope to set an example for new studies.

Keywords: lenalidomide, multiple myeloma, P-glycoprotein, verapamil

How to cite this article:
Tastemur S, Şencan M, Terzi H, Ergül M, Taştemur M. In vitro comparison of the cytotoxic effects of lenalidomide alone and in combination with verapamil on myeloma cell line. Egypt J Haematol 2021;46:185-91
How to cite this URL:
Tastemur S, Şencan M, Terzi H, Ergül M, Taştemur M. In vitro comparison of the cytotoxic effects of lenalidomide alone and in combination with verapamil on myeloma cell line. Egypt J Haematol [serial online] 2021 [cited 2022 May 14];46:185-91. Available from: http://www.ehj.eg.net/text.asp?2021/46/3/185/345245   Background 

Multiple myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells originating from B cells in the bone marrow. There is increased production of immunoglobulins (Igs) and light chains in MM. They are proteins that are detected by electrophoresis method. Overall, 60% of patients have IgG type M protein.

MM constitutes 1% of all cancers and 10–15% of hematological malignancies [1]. The average age at the time of diagnosis in MM is 70 years, and it is rare under the age of 40 [2]. It is more common in men than in women. Its annual incidence is 4–6/100 000 [3].

The diagnosis of MM is made by considering the diagnostic criteria revised by the International Myeloma Working Group in 2014 and shaped according to the results of bone marrow examination, radiography, and laboratory examinations [4].

There are both environmental factors such as radiation, dyes, petroleum products, aflatoxin, and pesticides, and cytogenetic factors like translocations and hyperdiploidy originating from the tumor itself [1].

Treatment options are basically high-dose chemotherapy and stem cell transplantation. Particularly, new agents are valuable because they significantly prolong progression-free survival [5]. However, MM still retains its feature of being a malignancy without a cure. The immunomodulatory drugs such as lenalidomide and pomalidomide; the proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib; CD8 monoclonal antibody daratumumab; and signaling lymphocytic activation molecule family 7 antibody elotuzumab have been studied much today and have significantly increased the treatment response compared with the conventional drugs used before. Nevertheless, drug resistance, which develops against chemotherapeutics, is an important problem in MM, as in many cancer types. The most important event accused in the pathogenesis of drug resistance in malignancies is the overexpression of P-glycoprotein (P-gp), which is a group of ATP-dependent carrier proteins (ABC proteins; ATP-binding cassette superfamily transporters). P-gp is an efflux protein responsible for the detoxification process in cells. In malignant cells, the excess P-gp gene expression creates drug resistance by preventing chemotherapeutics from penetrating into the cell and accumulating [6]. P-gp can be found in normal tissues such as blood–brain barrier, intestines, hepatocytes, kidneys, placenta, ovaries, and testicular tissue. Its physiological mission is to prevent the accumulation of toxic substances in tissues [7],[8]. It was observed that P-gp gene expression increased in 50% of patients with MM after chemotherapy [6].

The success of cancer chemotherapy depends on the concentration of the drug in the cell. As P-gp expression proteins prevent accumulation of these drugs, as a way of success in cancer treatment, the idea of suppressing this blockage of accumulation has gained importance.

In many studies, noncytotoxic chemosensitizing agents, which suppress the overexpression of P-gp, were combined with various chemotherapeutic agents to investigate whether they contribute to cytotoxicity. The most researched P-gp inhibitors are verapamil, cyclosporin-A, tamoxifen, and quinidine analogs [9],[10]. The antiarrythmic, antianginal, and antihypertensive calcium channel blocker verapamil is the first noncytotoxic P-gp inhibitor used for this purpose [11].

Many chemotherapeutic agents used in MM are substrates for P-gp. Lenalidomide is a weak substrate for P-gp. Nevertheless, it has been investigated whether verapamil and some other chemosensitizer agents will contribute to the transport or accumulation of lenalidomide via P-gp, and different results have been obtained [12],[13].

In our study, we aimed to evaluate the cytotoxicity increasing effect of verapamil on lenalidomide.

  Materials and methods 

The MM (U266) cell line obtained from the human peripheral blood used in the study was procured from the American Type Cell Collection.

Bilser Laminar Flow Cabinet inner surface was disinfected with 70% alcohol before each use. Cells stored frozen at −80°C were kept in a 37°C water bath for thawing. Dulbecco’s modification of eagle medium (DMEM) was brought to room temperature in a 37°C water bath in a mixture of molten cell and fetal bovine serum, which was taken into a 15-ml flask.

After centrifugation at 82 gravity for 5 min, the supernatant was carefully removed. The pellet was resuspended with 2 ml of medium and placed in a 25 cm2 flask. Overall, 10 ml of medium mixture, consisting of 89% Dulbecco’s modified eagle medium, 10% fetal bovine serum, and 1% Penicillin-Streptomycin, was added to the flask.

The cell line was placed in an incubator medium that provides 5% CO2 at 37°C to ensure cell proliferation. To maintain the cell viability and proliferation rate, medium centrifugation was performed at regular intervals to resuspend the pellet. Clustering of cells was prevented by intermittent washing with phosphate buffered saline. Controls were made against the possibility of contamination. When the cells reached 70% density, they were propagated by passaging. The cells resuspended in 1 ml of Roswell Park Memorial Institute culture medium were divided and placed in two separate 25 cm2 flasks, and 10 ml freshly prepared medium was added. Suspended cells, which were kept in the incubator, were then resuspended by centrifugation with adding 1 ml of Roswell Park Memorial Institute to the pellet. From this mixture, 1 : 100 dilution was made, and 10 µl of sample was taken into the Thoma slide and cell count was performed. The number of cells (n) per 16 squares on the Thoma slide was determined, and the total number of cells we had was calculated using the formula ‘n×104×dilution factor’. Overall, 10 000 cells per well were planted in a 96-well microplate.

After lenalidomide was dissolved in dimethyl sulfoxide and the stock solution was prepared, this solution was passed through a 0.2-µm syringe tip filter and sterilized (dimethyl sulfoxide ratio at the highest concentration 0.01%). Different concentrations (0.001, 0,01, 0.1, 1, 10, 50, and 100 µM) of lenalidomide to be applied to the cells were prepared by making serial dilutions from this stock solution. IC50 values were determined by applying lenalidomide in concentrations ranging from low to high (0.001, 0.01, 0.1, 1, 10, 50, 100 µM) to the U266 cell line. Moreover, the IC50 values of lenalidomide at the same concentrations were determined 1 h after adding 2.5 µg/ml verapamil. After the application of drugs, the microplate was left to incubate for 24 and 48 h.

The effect of lenalidomide in the combination with verapamil on cell viability was investigated by XTT test. The method of this test is based on the principle that metabolically active cells reduce XTT which is a tetrazolium salt, to orange formazan components, thanks to mitochondrial dehydrogenase enzymes. Although the formed dye is water soluble, the density of the dye can be read at the wavelengths (450 nm) given with a spectrophotometer.

Dye density (orange color) is proportional to the number of metabolically active cells. During the 24th and 48th hours of incubation, 50 µl of XTT solution was added to the wells with 1×104 cells each and incubated in the oven containing CO2 for 4 h.

At the end of the incubation period, the optical density value was read 450 nm in the microplate reader, and the cell viability rate of the control group was accepted as 100%, and it was calculated as follows: (concentration OD/control OD)×100. Applications were carried out six times.

Statistical analysis

The statistical evaluation of the experimental data was made with the “IBM SPSS statistics 24 programme”. While making statistical evaluations, results obtained from at least six independent trials were used. One-way analysis of variance (post-hoc Tukey) test was used to compare the differences between the groups. P value less than 0.05 value was accepted as statistically significant.

  Results 

Cytotoxic effect of lenalidomide on U266 cell line

Lenalidomide was incubated on the U266 cell line in increasing concentrations of 0.001, 0.01, 0.1, 1, 10, 50, and 100 µM, and at the end of 24 and 48 h, the IC50 for lenalidomide was calculated as 36 µM for 24 h and 26.6 µM for 48 h ([Figure 1]). The results were significant for 10, 50, and 100 µM concentrations (P<0.05).

  Cytotoxic effect of verapamil and lenalidomide combination on U266 cell line 

Verapamil was applied to MM cells at a concentration of 2.5 µg/ml, 1 h before the addition of lenalidomide. When lenalidomide was added and the incubation times of 24 and 48 h were completed, a significant cytotoxic effect was observed for the concentrations of 10, 50, and 100 µM again (P<0.05). The IC50 value calculated for lenalidomide after this combination application was 25.2 µM for 24 h and 21.7 for 48 h ([Figure 2]). Although it does not seem like a huge difference, the decrease in the IC50 was statistically significant.

Figure 2 Cytotoxic effect of verapamil and lenalidomide combination on U266 cell line.

Click here to view

Comparison of cytotoxic effects of lenalidomide alone and lenalidomide/verapamil combination on U266 cell line

Increasing concentrations of lenalidomide alone showed statistically significant cytotoxic effects at concentrations of 10 µM and above (P<0.05). Verapamil-applied MM cells were kept for 1 h in the incubator, and then the same concentrations of lenalidomide were applied, and at the end of the 24th hour and 48th hour, statistically significant cytotoxic effects were obtained at concentrations of 10 µM and above (P<0.05). The IC50 value of the verapamil/ lenalidomide combination was found to be significantly lower (36 and 25.2 µM for 24 h, and 26.6 and 21.7 µM for 48 h) compared with lenalidomide administration alone ([Figure 3]).

Figure 3 Comparison of cytotoxic effects of lenalidomide alone and lenalidomide/verapamil combination on U266 cell line.

Click here to view

  Discussion 

P-gp expression was found very low in untreated patients with MM. This shows that the accumulated drug dose increased P-gp expression [14]. Higher multidrug resistant (MDR) protein expression was observed in relapsed disease, indicating that treatment with cytarabine and vinblastine in leukemia increased expression of MDR proteins [15]. There are studies showing that these cells are more resistant to treatment [16]. Although the efflux rate of lenalidomide in MDCK-II cells (canis, distal tubular epithelium-like cells) was 1.9, the rate was found to be high as 3.6 in MDCK-II/P-gp cells with P-gp overexpression [17]. This again reveals the relationship between P-gp mediated drug efflux and resistance to lenalidomide. Another study in 1996 with doxorubicin and verapamil on the MM cell line suggested that verapamil was resistant to the P-gp suppression effect. In these resistant MM cells, membrane-bound P-gp expression was 40% less than in other MM cells [18].

In a 2012 study, although there was no substrate for P-gp, bortezomib reversed resistance with a different mechanism using the P-gp inhibitor verapamil. According to the results of this study with bortezomib and verapamil in four different mantle cell lymphoma cell lines, which are also resistant to bortezomib, verapamil significantly increased the cytotoxic effect of bortezomib. In the study, 240 µM of verapamil and 10 nM of bortezomib were added to the cell cultures. The level of inflammatory cytokines associated with the NF-KB pathway, such as interleukin-6 and interleukin-8, was found to be significantly lower after adding verapamil [19].

Although it was made with a chemotherapeutic agent that has cytotoxic effect with a different mechanism such as bortezomib, this study showed the contribution of verapamil to cytotoxicity on a hematological origin malignancy is very important in terms of guiding us.

There are many studies on lenalidomide resistance with different P-gp inhibitors. Some have no significant results, whereas others are very promising. In a phase I study with no significant results, 300–600 mg twice daily quinidine and 25 mg temsirolimus combinations were evaluated for their pharmacokinetic interactions in patients receiving 25 mg of lenalidomide, but no relevance was found [20]. Another study revealed that itraconazole increased the intracellular level by decreasing the excretion of lenalidomide as a P-gp inhibitor [21].

Partial benefit was generally observed in studies with verapamil. As an important reason for this, it is shown that the concentration required to completely eliminate the drug resistance is approximately ten times the dose that produces atrioventricular block in human [22]. To prevent this cardiotoxic effect, researchers have suggested that it is convenient to use several chemosensitizing agents by combining them, and that anti-MDR efficacy can also be increased [23]. In 2014, it was suggested that verapamil was well tolerated and safe in addition to its contribution to cytotoxicity in a study performed on doxorubicin-resistant murine breast cancer cells on mouse models with verapamil and doxorubicin [24].

In a phase I/II study, verapamil was implemented with vincristine/doxorubicin/dexamethasone (VAD) as an MDR modulator in patients with refractory MM. A 50% partial response rate has been achieved, but it has been decided at the end of the study that it cannot be used as a treatment protocol owing to the excessive toxicity [11],[25]. In a phase III clinical study, it was revealed that its use was restricted due to similar adverse effects despite the lower doses of verapamil [26],[27]. In another study, the effect of verapamil was investigated in vitro in reducing the resistance to doxorubicin and vincristine in bone marrows obtained from 59 patients with MM, and it was revealed that verapamil significantly increased the sensitivity to chemotherapeutics. In the clinical part of the study, 22 patients were administered high-dose (295 ng/ml) verapamil infusion in addition to the VAD regimen. A significant elongation was observed in the overall survival time. As a result of the study, it has been suggested that verapamil both reduces the present resistance and decreases the development of new resistance when applied in the initial treatment before it develops [28]. This is a promising result in terms of preventing resistance development by combining verapamil with appropriate regimens not only in refractory/relapsed MM treatment but also in primary treatment and deserves further investigation.

In a 2014 study conducted by Abraham et al. [29], it was found that drug intake was significantly higher in patients who received doxorubicin with P-gp inhibitors such as verapamil and psorospermin, and their sensitivity to doxorubicin increased.

In 2017, in a study with verapamil administration to docetaxel and vinblastine in the H1299 lung cancer cell line, it was observed that P-gp levels increased six-fold and two-fold, respectively, 24 h after administration of docetaxel and vinblastine separately. The combination obtained with the addition of verapamil has been shown to lower this increased level of P-gp. As a result of the study, it was found that verapamil increased the effectiveness of the chemotherapy and decreased the drug resistance [30].

In a study assessing the contribution of the antitumor agent called SJG-136 to the efficacy of verapamil by P-gp suppression in the colon cancer cell line, cells received 5 µg/ml verapamil 1 h before the administration of SJG-136, and at the end of the study, the IC50 value decrease from 3.7 to 0.3 nM [31].

Based on these data, we aimed to increase the cytotoxic effectiveness of lenalidomide in our study and applied verapamil 1 h before lenalidomide. We had a decrease in IC50 value from 36 to 25.2 µM after 24 h. Both the study with SJG-136 and our study show that giving verapamil 1 h before the cytotoxic agent (lenalidomide for our study) would be more suitable for increasing efficacy.

In our study, we showed that administration of 2.5 µg/ml of verapamil 1 h before the application of lenalidomide significantly increased the cytotoxic effect of lenalidomide and significantly decreased the IC50 value at 10, 50, and 100 µM concentrations of lenalidomide (P<0.05).

Suppression of P-gp − the most important mechanism of drug resistance development against lenalidomide-via verapamil significantly decreased the IC50 value of lenalidomide. This decrease in IC50 means a decrease in the effective dose of lenalidomide and adverse effects that may occur during treatment. As a result, in this study, we assessed the effect on MM cell lines, where an increase in cytotoxic effect was observed with the addition of verapamil to lenalidomide.

  Conclusion 

Despite many recent developments, problems regarding MM treatment and resistance still remain as an important problem. Today, increasing the effectiveness of drugs developed for treatment and preventing resistance mechanisms are important. Chemotherapeutic drug delivery via P-gp is an important part of these mechanisms. Therefore, in our study, we aimed to reveal the increase in cytotoxic effect of lenalidomide, which has an important place in MM treatment, with the contribution of a cheap and easily available preparation such as verapamil. The negative effect on the heart rate, which is the reason why the success in in-vitro studies could not be achieved in clinical studies, seems to be an important dilemma, and more preclinical and clinical studies with verapamil are needed to overcome this problem. We hope that our study will be an example for the research studies to be conducted.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

  References 
1.Shaheen SP, Talwalkar SS, Medeiros LJ. Multiple myeloma and immunosecretory disorders: an update. Adv Anat Pathol 2008; 15:196–210.  
    2.Jameson JL, Fauci AS, Kasper DL, Hauser SL, Longo DL, Loscalzo J. Harrison’s principles of internal medicine (2018) 20th Edition. Part 4 Oncology and Hematology, Section 2, Hematopoetic Disorders ISBN 9781259644030.  
    3.Harousseau JL, Moreau P. Autologous hematopoietic stem-cell transplantation for multiple myeloma. N Engl J Med 2009; 360:2645–2654.  
    4.Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia 2009; 23:3–9.  
    5.Wintrobe MM, Greer JP. Wintrobe’s clinical hematology. 11th ed. Philadelphia: Lippincott Williams & Wilkins; 2004. p. 2555–2564.  
    6.Schwarzenbach H. Expression of MDR1/P-glycoprotein, the multidrug resistance protein MRP, and the lung-resistance protein LRP in multiple myeloma. Med Oncol 2002; 19:87–104.  
    7.Pace L, Catalano L, Del Vecchio S, De Renzo A, Fonti R, Salvatore Bet al. Washout of [99mTc] sestamibi in predicting response to chemotherapy in patients with multiple myeloma. Q J Nucl Med Mol Imaging 2005; 49:281–285.  
    8.Lin B. Patupilone (epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo. Blood 2005; 105:350–357.  
    9.Lehnert M, Dalton WS, Roe D, Emerson S, Salmon SE. Synergistic inhibition by verapamil and quinine of P-glycoprotein-mediated multidrug resistance in a human myeloma cell line model. Blood 1991; 77:348–354.  
    10.Ford JM. Modulators of multidrug resistance: preclinical studies. Hematol Oncol Clin North Am 1995; 9:337–362.  
    11.Fisher GA, Lum BL, Hausdorff J, Sikic BI. Pharmacological considerations in the modulation of multidrug resistance. Eur J Cancer 1996; 32A:1082–1088.  
    12.Chen N, Weiss D, Reyes J, Liu L, Kasserra C, Wang Xet al. No clinically significant drug interactions between lenalidomide and P-glycoprotein substrates and inhibitors: results from controlled phase I studies in healthy volunteers. Cancer Chemother Pharmacol 2014; 73:1031–1039.  
    13.Takahashi N, Miura M, Kameoka Y, Abumiya M, Sawada K. Drug interaction between lenalidomide and itraconazole. Am J Hematol 2011; 87:338–339.  
    14.Linsenmeyer ME, Jefferson S, Wolf M, Matthews JP, Board PG, Woodcock DM. Levels of expression of the mdr1 gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma. Br J Cancer 1992; 65:471–475.  
    15.Xu YC, Lin YM, Zhang FC. The relationship between abnormal MDR gene expression and chemotherapy response in lymphoid malignancies. Zhonghua Zhong liu za zhi 2006; 28:353–356.  
    16.Yeh JJ, Hsu WH, Wang JJ, Ho ST, Kao A. Predicting chemotherapy response to paclitaxel-based therapy in advanced non-small-cell lung cancer with P-glycoprotein expression. Respiration 2003; 70:32–35.  
    17.Weber DM, Chen C, Niesvizky R, Wang M, Belch A, Stadtmauer EAet al. Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America. N Engl J Med 2007; 357:2133–2142.  
    18.Abbaszadegan MR, Foley NE, Gleason-Guzman MC, Dalton WS. Resistance to the chemosensitizer verapamil in a multi-drug-resistant (MDR) human multiple myeloma cell line. Int J Cancer 1996; 66:506–514.  
    19.Chen Z, Romaguera J, Wang M, Fayad L, Kwak LW, McCarty N. Verapamil synergistically enhances cytotoxicity of bortezomib in mantle cell lymphoma via induction of reactive oxygen species production. Br J Haematol 2012; 159:243–246.  
    20.Safa AR. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil. Proc Natl Acad Sci USA 1988; 85:7187–7191.  
    21.Hofmeister CC, Yang X, Pichiorri Fet al. Phase I trial of lenalidomide and CCI-779 in patients with relapsed multiple myeloma: evidence for lenalidomide-CCI-779 interaction via P-glycoprotein. J Clin Oncol 2011; 29:3427–3434.  
    22.Krishna R, McIntosh N, Riggs KW, Mayer LD. Doxorubicin encapsulated in sterically stabilized liposomes exhibits renal and biliary clearance properties that are independent of valspodar (PSC 833) under conditions that significantly inhibit nonencapsulated drug excretion. Clin Cancer Res 1999; 5:2939–2947.  
    23.Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999; 39:361–398.  
    24.McCarthy M, Auda G, Agrawal S, Taylor A, Backstrom Z, Mondal D, Moroz K, Dash S. In vivo anticancer synergy mechanism of doxorubicin and verapamil combination treatment is impaired in BALB/c mice with metastatic breast cancer. Exp Mol Pathol 2014; 97:6–15.  
    25.Robak P, Drozdz I, Szemraj J, Robak T. Drug resistance in multiple myeloma. Cancer Treat Rev 2018; 70:199–208.  
    26.Dalton WS, Crowley JJ, Salmon SS, Grogan TM, Laufman LR, Weiss GR, Bonnet JD. A phase III randomized study of oral verapamil as a chemosensitizer to reverse drug resistance in patients with refractory myeloma. A southwest oncology group study. Cancer 1995; 75:815–820.  
    27.Yang HH, Ma MH, Vescio RA, Berenson JR. Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis. J Clin Oncol 2003; 21:4239–4247.  
    28.Salmon SE, Dalton WS, Grogan TM, Plezia P, Lehnert M, Roe DJ, Miller TP. Multidrug-resistant myeloma: laboratory and clinical effects of verapamil as a chemosensitizer. Blood 1991; 78:44–50.  
    29.Abraham J, Salama NN, Azab AK. The role of P-glycoprotein in drug resistance in multiple myeloma. Leuk Lymphoma 2015; 56:26–33.  
    30.Jaferian S, Soleymaninejad M, Daraee H. Verapamil (VER) enhances the cytotoxic effects of docetaxel and vinblastine combined therapy against non-small cell lung cancer cell lines. Drug Res (Stuttg) 2018; 68:146–152.  
    31.Guichard SM, Macpherson JS, Thurston DE, Jodrell DI. Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136. Eur J Cancer 2005; 41:1811–1818.  
    
  [Figure 1], [Figure 2], [Figure 3]