Combined antitumoral effects of pretubulysin and methotrexate

Abstract Pretubulysin (PT), a potent tubulin‐binding antitumoral drug, and the well‐established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI‐nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT‐triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F‐actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.

are used widely in the clinics. By either stabilizing or destabilizing microtubules, they lead to a disruption of the microtubule network and to G2/M arrest. 1 However, given the development of resistances that for instance frequently occur with Vinca alkaloids, the need for new drugs of this class becomes crucial. 2,3 Tubulysins, a family of natural compounds of myxobacterial origin, are a powerful and highly effective therapeutic group. By binding to the vinca domain of β-tubulin, tubulysins prevent tubulin polymerization which ultimately results in microtubule depletion and apoptosis of the treated cells. 4,5 Pretubulysin (PT) is a biosynthetic precursor of the tubulysins and better accessible by chemical synthesis. 6,7 Moreover, it displays remarkable antitumoral potency in the subnanomolar region. 6,8 PT not only leads to reduced tumor cell growth of different cell lines 6 and inhibits cancer cell migration in vitro, 8 it also shows great potential in vivo: PT inhibits tumor growth 6,[8][9][10][11] and metastasis 6,12 and also significantly reduces angiogenesis. 8,10 Antifolates, which belong to the class of antimetabolites, were among the first chemotherapeutic drugs to be investigated for the cure of metastatic cancer. As folate antagonists, they make use of the reduced folate carrier (RFC) or the folate receptor (FR) to enter the cell. [13][14][15] The FR is overexpressed in many epithelial tumors and therefore plays an important role in targeted cancer therapy. 16,17 Methotrexate (MTX) is the most prominent representative of this group. After successfully entering the cell, it competitively inhibits the dihydrofolate reductase (DHFR) and by that the conversion of folic acid to dihydrofolic acid and tetrahydrofolic acid. These are required for the biosynthesis of purines, thus the de novo synthesis of DNA. 18 However, acquired resistance to MTX represents a common problem of monotherapy approaches. [19][20][21] This hurdle can possibly be overcome by combining MTX with a second antitumoral agent.
Combination therapy is an approach that combines two or more therapeutic agents in order to address several targets, possibly reduce resistance formation and increase the therapeutic efficacy while potentially decreasing dosages. 22,23 Already in 1965, a combination chemotherapy approach referred to as POMP regimen was successfully administered. Apart from MTX, it contained 6-mercaptopurine, vincristine, and prednisone and resulted in long-term remission in children with acute lymphocytic leukemia. 24,25 In a previous experiment, in which the novel tubulin inhibitor PT was conjugated with MTX-containing oligomers for FR-targeted delivery, we searched for a possible combination effect. 11 Hence, this drug combination was chosen in this study to further boost the encouragingly high potency of PT. The combination of both drugs was analyzed in terms of cytotoxicity, apoptosis, its influence on the tumor cell cycle and the cytoskeleton. Moreover, in vivo antitumor activity of the combination approach was evaluated in a treatment experiment in two tumor mouse models. All experiments were performed in L1210 leukemia and KB cervix carcinoma cells since both are sensitive to PT and MTX. Furthermore, MTX has been well established for the treatment of leukemia.

Cell viability assay (MTT)
L1210 suspension cells were seeded at a density of 5 × 10 3 cells per well (96-well plate) in 80 μL growth medium 4 hours prior to treatment. Twenty microliters of HBG, PT, MTX, or PT+MTX in HBG were added and plates were left to incubate for 72 hours. Ten microliters of MTT (5 mg mL −1 ) were added to each well, and incubated for 2 hours in a cell culture incubator. For cell lysis, a solution of 10% sodium dodecyl sulfate (SDS) in 0.01 mol L −1 hydrochloric acid (HCl) was added and incubated overnight before photometric analysis.
KB cells were seeded at a density of 2.5 × 10 3 cells per well in 100 μL of growth medium 24 hours prior to treatment. Medium was changed 1 hour before treatment. Twenty microliters of HBG, PT, MTX, or PT+MTX in HBG were added, and plates were left to incubate for 72 hour. Ten microliters of MTT (5 mg mL −1 ) were added to each well and incubated for 2 hours in a cell culture incubator.
After formazan formation, medium and MTT were removed, and cells were frozen at −80°C. After at least 30 minutes in the freezer, formazan was dissolved in 100 μL of DMSO. Absorption was measured at a wavelength of 590 nm against a reference wavelength of 630 nm using a SpectraFluor ™ Plus microplate reader (Tecan, Groedig, Austria). Cell viability was calculated as percentage of absorption compared to wells treated with HBG only. All experiments were performed in quintuplicates.

| In vivo experiments
Murine leukemia tumor model

| In vitro antitumoral activity of PT, MTX or PT+MTX
L1210 and KB cells were treated with PT and MTX for 72 hours at a set drug molar ratio of 1 to 3, and cell viability of drug-treated cells was determined by MTT assay (Figure 1). In case of L1210 cells ( Figure 1A (see Figure S1).

| Confocal laser scanning microscopy of drugtreated cells
The effects of PT, MTX, and PT+MTX on the DNA, the actin cytoskeleton and on the microtubules of L1210 and KB cells were determined by confocal laser scanning microscopy (CLSM). Figure 4 depicts the disruption of the microtubule network of L1210 and KB cells caused by PT treatment, already after 24 hours treatment (see also Figure S5 for nonmerged images). L1210 cells have lost their structural integrity, the microtubule network seems to be located extracellularly. PT also induces nuclear fragmentation of L1210 cell nuclei. Moreover, a change in the F-actin cytoskeleton upon MTX treatment can be seen especially for KB cells ( Figure 4B

F I G U R E 1 Combination effect of pretubulysin (PT) and methotrexate (MTX) on cultured L1210 cells but not KB cells. Cell viability and IC50 values of drug-treated (A) L1210 cells and (B) KB cells. Cell viability was measured with an MTT assay after 72 hours
treatment and is presented as the mean + SD (n = 5) in % relative to buffer (HEPES buffered glucose) treated cells. c (nmol L −1 ) refers to the concentration of PT, the concentration is 3-fold higher for MTX, due to the 1:3 molar drug ratio (**P < 0.01; ***P < 0.001; ****P < 0.0001)  After the treatment start, tumor growth in the HBG-injected group proceeded rapidly, so the first animal had to be sacrificed after 9 days. Tumor growth in MTX-treated animals was slightly slowed down after 1 week of treatments and the first animal was sacrificed after 12 days. Notably, tumor growth in both PT containing groups was retarded from the first injection on and could be further inhibited in the PT+MTX combination group ( Figure 6A). While the first animal of the PT group had to be sacrificed on day 15 after treatment start, PT+MTX combination led to a survival of all animals until day 21. Figure 6B  The weight of animals was monitored regularly; the unobtrusive weight development in all groups indicates that treatments were well tolerated by the animals (Figure S6B). tetrahydrofolate and thus, the C1 metabolism. As a result, no nucleotides are synthesized which would be essential for DNA synthesis.
Furthermore, PT does not have to be converted into its active form before carrying out its toxic effect. The earlier onset in toxicity leads to a G2/M arrest, and arrested cells cannot in turn be affected by MTX anymore.
With regard to drug-induced apoptotic events in L1210 or KB cells, treatment with PT or MTX alone resulted in a steady increase of apoptotic cells which is in accordance with previous work. 6,10,34,37 Yet, apoptosis could not be further enhanced by combining both agents.
Previous studies show PT-induced depolymerization of microtubules in different cell lines, 6 In contrast to cell culture cytotoxicity, a lack of antitumoral activity of MTX was observed in vivo at the applied dosage of 5 mg kg −1 , which is supported by previous studies. 45,46 The dosage of MTX used in the mouse experiments was based on efficacy in cell culture.
Burger et al described 100 mg kg −1 as the maximum tolerated dose of MTX for NMRI-nude mice. 47 In our additional MTX dose finding experiments, even after multiple injections with the highest tolerated dose of 80 mg kg −1 , we did not observe any significant L1210 tumor growth inhibition in vivo. An acquired chemoresistance against MTX can be excluded, since the MTX-treated tumors are still MTX sensitive in cell culture. On the other hand, PT at the well-tolerated 2 mg kg −1 dose exhibited a clear antitumoral effect on L1210 tumors in vivo. This is also in accordance with our recent work in combining PT with antitumoral EG5 siRNA. 9 Encouragingly, this favorable PT effect could be further enhanced by the co-administration of the rather low dose of 5 mg kg −1 MTX, resulting in a significantly retarded tumor growth in the combination group. The boosting effect of low dose MTX is remarkable, considering the lack of antitumoral effects of the single drug at even 20-fold higher dose.
In the KB human cervix carcinoma tumor model, PT had already previously demonstrated antitumoral effects. 11 In this study, the antitumoral activity of 2 mg kg −1 PT could be confirmed, while MTX only slightly inhibited KB tumor growth. This is consistent with the known in vitro chemoresistance of KB cells to MTX 48 and in vivo studies. 46 Importantly, also in this carcinoma model, the co-administration of