Effects of mTOR inhibitors and cytoskeletal-directed agents alone and in combination against normal and neoplastic hematopoietic cells in vitro.

The mechanistic target of rapamycin (mTOR) controls cell growth and enlargement and has been found to be aberrant in a wide variety of malignancies. Although mTOR is already an attractive antineoplastic target, overexpression or aberrant expression of mTOR may also provide an opportunity to further increase the size differential between malignant and normal cells, providing an opportunity to amplify and exploit cell size differences between neoplastic cells and their normal counterparts using physiochemical treatment modalities. Therefore, this study sought to quantify the concentration response and time course effects of rapamycin on cell cycle entry, cell enlargement, and cell proliferation in U937 human monocytic leukemia and human hematopoietic stem cells (hHSCs). In addition, the effects of combination treatment with mTOR inhibitors (rapamycin, everolimus, and temsirolimus) and cytoskeletal-directed agents (cytochalasin B and vincristine) in leukemic cells (U937, THP1, K562, Molt-4, and L1210) were assessed for potential drug synergy. While both U937 cells and hHSCs exhibited a marked reduction in cell volume, U937 cells were able to proliferate in the presence of rapamycin ranging from 0.5 nM to 10 µM (10,000 nM), whereas hHSCs were able to proliferate only at lower concentrations, and were completely inhibited from proliferation by 8 nM rapamycin. These effects were observed with as little as 0.5 nM rapamycin, demonstrating the profound affinity the compound has for FK-binding protein 12 (FKBP12), which subsequently forms the FKBP12/rapamycin complex to inhibit mTOR. Rapamycin continued to exert effects on cell size and proliferation even at 10 µM, without producing marked cytotoxicity. Although cytochalasin B and vincristine were unable to substantially enlarge rapamycin-treated leukemia cells, it appears that rapamycin and its associated analogs everolimus and temsirolimus have notable synergistic potential with microfilament-disrupting cytochalasin B and microtubule-disrupting vincristine as assessed by comparative effects on cell growth, annexin V staining, IC30 isobolograms, and Chou-Talalay statistics. These observations indicate a potentially novel therapeutic rationale for hematological malignancies and for other cancers to elicit the preferential destruction of neoplastic cells that aberrantly express mTOR.

Effects of cytochalasin congeners, microtubule-directed agents, and doxorubicin alone or in combination against human ovarian carcinoma cell lines in vitro.

BACKGROUND: Although the actin cytoskeleton is vital for carcinogenesis and subsequent pathology, no microfilament-directed agent has been approved for cancer chemotherapy. One of the most studied classes of microfilament-directed agents has been the cytochalasins, mycotoxins known to disrupt the formation of actin polymers. In the present study, we sought to determine the effects of cytochalasin congeners toward human drug sensitive and multidrug resistant cell lines. METHODS: SKOV3 human ovarian carcinoma and several multidrug resistant derivatives were tested for sensitivity against a panel of nine cytochalasin congeners, as well as three clinically approved chemotherapeutic agents (doxorubicin, paclitaxel, and vinblastine). In addition, verapamil, a calcium ion channel blocker known to reverse P-glycoprotein (P-gp) mediated drug resistance, was used in combination with multiple cytochalasin congeners to determine whether drug sensitivity could be increased. RESULTS: While multidrug resistant SKVLB1 had increased drug tolerance (was more resistant) to most cytochalasin congeners in comparison to drug sensitive SKOV3, the level of resistance was 10 to 1000-fold less for the cytochalasins than for any of the clinically approved agents. While cytochalasins did not appear to alter the expression of ATP binding cassette (ABC) transporters, several cytochalasins appeared to inhibit the activity of ABC transporter-mediated efflux of rhodamine 123 (Rh123), suggesting that these congeners do have affinity for drug efflux pumps. Cytochalasins also appeared to significantly decrease the F/G-actin ratio in both drug sensitive and drug resistant cells, indicative of marked microfilament inhibition. The cytotoxicity of most cytochalasin congeners could be increased with the addition of verapamil, and the drug sensitivity of resistant SKVLB1 to the clinically approved antineoplastic agents could be increased with the addition of cytochalasins. As assessed by isobolographic analysis and Chou-Talalay statistics, cytochalasin B and 21,22-dihydrocytochalasin B (DiHCB) demonstrated notable synergy with doxorubicin and paclitaxel, warranting further investigation in a tumor-bearing mammalian model. CONCLUSION: Cytochalasins appear to inhibit the activity of P-gp and potentially other ABC transporters, and may have novel activity against multidrug resistant neoplastic cells that overexpress drug efflux proteins.

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells.

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.

Preparation, In Vivo Administration, Dose-Limiting Toxicities, and Antineoplastic Activity of Cytochalasin B.

An effective and inexpensive protocol for producing cytochalasins A and B is being disclosed to propose a viable method by which to examine the in vivo antineoplastic activity of these congeners in preclinical tumor-bearing mammalian models. In addition, we determine the maximum tolerated doses of cytochalasin B using multiple routes and formulations, characterize the tissue distribution of intravenous bolus cytochalasin B, and assess the in vivo antineoplastic activity of cytochalasin B in comparison in doxorubicin in Balb/c mice challenged intradermally with M109 murine lung carcinoma. We also examine the effects of cytochalasin B against several other murine neoplastic cell lines (Lewis lung, LA4, B16F10, and M5076). Finally, we examine a potential mechanism of the antimetastatic activity of cytochalasin B by observing the effects of the agent on the secretion of N-acetylglucosaminidase (GlcNACase) by B16BL6 and B16F10 murine melanomas in vitro. The results of the study can be summarized as follows: 1) Cytochalasin B can be safely administered intravenously, intraperitoneally, and subcutaneously in murine models, with the maximum tolerated dose of all routes of administration being increased by liposome encapsulation. 2) Cytochalasin B can significantly inhibit the growth of tumors in mice challenged with M109, Lewis lung, LA4, B16F10, or M5076, producing long-term survival against lung carcinomas and adenocarcinomas (M109, Lewis lung, and LA4) and B16F10 melanoma, but not M5076 sarcoma. These effects were comparable to intraperitoneally administered doxorubicin. 4) Low concentrations of cytochalasin B inhibit the secretion of GlcNACase, indicating that cytochalasin B may inhibit metastatic progression by mechanisms not directly associated with its influence on cell adhesion and motility.

Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy.

BACKGROUND: Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. RESULTS: Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. CONCLUSION: Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

Effects of alkylation and immunopotentiation against Ehrlich ascites murine carcinoma in vivo using novel tetra-O-acetate haloacetamido carbohydrate analogs.

Tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) are novel alkylating agents that appear to have alkylating activity at the plasma membrane, specificity against neoplastic cells, and may potentiate host leukocyte influx. This study sought to characterize the chemical attributes and in vivo activity of Tet-OAHCs. Four Tet-OAHCs were assessed for their partition coefficient and alkylating activity to determine cellular environments where adduct formation would be favorable. In vitro, IC50 values of all four Tet-OAHCs were determined against Ehrlich ascites murine carcinoma, as well as two leukemias (U937 human monocytic leukemia and L1210 murine lymphoid leukemia) to assess their cytotoxicity in multiple neoplastic cell lines. In vivo, B6D2F1 and CD2F1 mice were challenged i.p. with Ehrlich ascites carcinoma prior to, or after being treated with a single dose of one of the analogs. Finally, a quantitative comparison of host leukocyte influx between Tet-OAHCs and other alkylating agents was performed to confirm previous in vivo observations that the tetra-O-acetate carbohydrate moiety is important for inducing a host leukocyte response in murine models. The results can be summarized as follows: 1) Tet-OAHCs appear to demonstrate high alkylating activity in amphiphilic environments. 2) All four congeners have comparable in vitro cytotoxicities against the neoplastic cell lines examined. 3) The analogs demonstrate marked in vivo activity in both B6D2F1 and CD2F1 mice challenged with a lethal dose of Ehrlich ascites carcinoma, and frequently produce long term survival at 60 days, which is not observed in simple halo derivatives or two currently approved antineoplastic agents (daunorubicin and mechlorethamine). These effects are observed when the agents are administered either before or after the tumor challenge. 4) The carbohydrate moiety appears to be important for potentiating host leukocyte influx, as Tet-OAHCs, but not other alkylating agents demonstrated such activity in vivo.

Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.

Sonodynamic therapy (SDT) is a form of ultrasound therapy that has been shown to preferentially damage malignant cells based on the relatively enlarged size and altered cytology of neoplastic cells in comparison to normal cells. This study sought to determine whether cytoskeletal-directed agents that either disrupt (cytochalasin B and vincristine) or rigidify (jasplakinolide and paclitaxel) microfilaments and microtubules, respectively, affect ultrasonic sensitivity. U937 human monocytic leukemia cell populations were treated with each cytoskeletal-directed agent alone, and then sonicated at 23.5 kHz under relatively low power and intensity (20-40 W; 10-20 W/cm(2)), or at 20 kHz using moderate power and intensity (60 W; 80 W/cm(2)). In addition, human leukemia lines U937, THP1, K562, and Molt-4, and the murine leukemia line L1210 were sonicated using pulsed 20 kHz ultrasound (80.6 W; 107.5 W/cm(2)) both with and without the addition of cytoskeletal-directed agents to assess whether cytoskeletal-directed agents can potentiate ultrasonic sensitivity in different leukemia lines. Human hematopoietic stem cells (hHSCs) and leukocytes were sonicated with continuous 23.5 kHz ultrasound (20 W; 10 W/cm(2)) to determine whether this approach elicited the preferential damage of neoplastic cells over normal blood components. To determine whether ultrasonic sensitivity is exclusively dependent on cell size, leukemia cells were also enlarged via alteration of cell growth parameters including serum deprivation and re-addition, and plateau-phase subculturing. Results indicated that cytochalasin B/ultrasound treatments had the highest rates of initial U937 cell damage. The cells enlarged and partially synchronized, either by serum deprivation and re-addition or by plateau-phase subculturing and synchronous release, were not comparably sensitive to ultrasonic destruction based solely on their cell size. In addition, cytochalasin B significantly potentiated the ultrasonic sensitivity of all neoplastic cell lines, but not in normal blood cells, suggesting that preferential damage is attainable with this treatment protocol. Therefore, it is likely that ultrasonic cell lysis depends not only on cell size and type, but also on the specific molecular mechanisms used to induce cell enlargement and their effects on cell integrity. This is supported by the fact that either the microfilament-or microtubule-disrupting agent produced a higher rate of lysis for cells of a given size than the corresponding stabilizing agents.

Chemotherapy in vivo against M109 murine lung carcinoma with cytochalasin B by localized, systemic, and liposomal administration.

Cytochalasin B is a potentially novel microfilament-directed chemotherapeutic agent that prevents actin polymerization, thereby inhibiting cytokinesis. Although cytochalasin B has been extensively studied in vitro, only limited data are available to assess its in vivo potential. Cytochalasin B was administered to Balb/c mice challenged i.d. with M109 murine lung carcinoma to determine whether the agent could affect an established i.d. tumor when the compound is administered s.c. in the region of the i.d. tumor, but not in direct contact with it. Cytochalasin B was also administered either i.p. or s.c. at a distant site or i.v. to determine whether it could affect the long-term development of an established i.d. tumor. Cytochalasin B was then liposome encapsulated to determine whether the maximum tolerated dose (MTD) of the compound could be increased, while reducing immunosuppression that we have previously characterized. Liposomal cytochalasin B was also administered to mice challenged i.d. with M109 lung carcinoma to assess its chemotherapeutic efficacy. The results can be summarized as follows: 1) cytochalasin B substantially delayed the growth of i.d. M109 tumor nodules, inhibited metastatic progression in surrounding tissues, and produced long-term cures in treated mice; 2) liposomal cytochalasin B increased the i.p. MTD by more than 3-fold, produced a different distribution in tissue concentrations, and displayed antitumor effects against M109 lung carcinoma similar to non-encapsulated cytochalasin B. These data show that cytochalasin B exploits unique chemotherapeutic mechanisms and is an effective antineoplastic agent in vivo in pre-clinical models, either in bolus form or after liposome encapsulation.

Chemotherapy with cytochalasin congeners in vitro and in vivo against murine models.

Background Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. One of the most studied microfilament-directed agents has been cytochalasin B, a mycogenic toxin known to disrupt the formation of actin polymers. Therefore, this study sought to expand on our previous work with the microfilament-directed agent, along with other less studied cytochalasin congeners. Materials and Methods We determined whether cytochalasin B exerted significant cytotoxic effects in vitro on adherent M109 lung carcinoma and B16BL6 and B16F10 murine melanomas, or on suspension P388/ADR murine leukemia cells. We also examined whether cytochalasin B, its reduced congener 21, 22-dihydrocytochalasin B (DiHCB), or cytochalasin D could synergize with doxorubicin (ADR) against ADR-resistant P388/ADR leukemia cells, and produce significant cytotoxicity in vitro. For in vivo characterization, cytochalasins B and D were administered intraperitoneally (i.p.) to Balb/c mice challenged with drug sensitive P388-S or multidrug resistant P388/ADR leukemias. Results Cytochalasin B demonstrated higher cytotoxicity against adherent lung carcinoma and melanoma cells than against suspension P388/ADR leukemia cells, as assessed by comparative effects on cell growth, and IC50 and IC80 values. Isobolographic analysis indicated that both cytochalasin B and DiHCB demonstrate considerable drug synergy with ADR against ADR-resistant P388/ADR leukemia, while cytochalasin D exhibits only additivity with ADR against the same cell line. In vivo, cytochalasins B and D substantially increased the life expectancy of mice challenged with P388/S and P388/ADR leukemias, and in some cases, produced long-term survival. Conclusion Taken together, it appears that cytochalasins have unique antineoplastic activity that could potentiate a novel class of chemotherapeutic agents.

Enlargement and multinucleation of u937 leukemia and MCF7 breast carcinoma cells by antineoplastic agents to enhance sensitivity to low frequency ultrasound and to DNA-directed anticancer agents.

BACKGROUND/AIM: Cytochalasin B is a mycogenic toxin that preferentially damages malignant cells through multiple mechanisms. The microfilament-disrupting agent inhibits cytokinesis, producing enlarged and multinucleated neoplastic cells without enlarging or producing multinucleated normal cells. In addition, cytochalasin B has been shown to induce apoptosis and to increase the mitochondrial activity of malignant cells. In spite of these pharmacological properties potentially exploitable in cancer chemotherapy, no cytochalasin congener or derivative and indeed no microfilament-directed agent has yet been examined in the clinic. Nevertheless, it will likely be necessary to combine microfilament-directed agents with other chemotherapeutic agents, and potentially with other anti-neoplastic modalities to amplify the mechanisms by which microfilament-directed agents inflict damage. These combinations could increase the likelihood of obtaining clinically useful activities with microfilament-directed agents and decrease the often inevitable emergence of drug resistance. Therefore, this study intends to determine appropriate chemotherapeutic agents to use concurrently with cytochalasin B and with other microfilament-directed agents. MATERIALS AND METHODS: Since cytochalasin B has shown in vitro efficacy against anchorage-independent growth, as well as against attached malignancies, both U937 human monocytic leukemia and MCF7 human breast carcinoma cells were evaluated. These cell lines were assessed for their sensitivity to a comprehensive array of chemotherapeutic agents that could amplify the cytoskeletal effects of microfilament-directed agents or that could themselves be potentiated by the cellular effects of such agents. In addition, clinically-approved microtubule-directed agents, as well as clinically-active anti-neoplastic agents not specifically cytoskeletal-directed, were examined for their ability to potentiate cell enlargement, one of the hallmark features of microfilament-directed agents. Conditions for inducing optimal enlargement and multinucleation of neoplastic cells with cytochalasin B were also defined. RESULTS: U937 and MCF7 cells have differing sensitivities to chemotherapeutic agents indicating that different regimens will likely be needed for various cell types in concomitant cytochalasin B-mediated chemotherapy. It was noted that microtubule-directed agents (paclitaxel and vincristine) would likely have a synergistic effect with cytochalasin B as they produced a substantial enlargement in viable cells at their 50% inhibitory (IC50) values. Interestingly, doxorubicin and mitomycin C also produced considerable cell enlargement, suggesting that nucleic acid-directed agents may be used to further enhance the cell-enlargement and multinucleation effects of microfilament-directed agents if appropriate sequences and concentrations can be found for the combination of agents. A subsequent publication in this series will examine the optimal combinations of chemotherapeutic agents with microfilament-directed agents in regards to drug concentrations and sequential timing. U937 cells exposed to cytochalasin B exhibited substantial cell enlargement and multinucleation that was still prevalent 8 days post-administration depending on the concentration used. CONCLUSION: Taken together, it appears that cytochalasin B has substantial synergistic potential with microtubule- and nucleic acid-directed agents.

Tolerated doses in zebrafish of cytochalasins and jasplakinolide for comparison with tolerated doses in mice in the evaluation of pre-clinical activity of microfilament-directed agents in tumor model systems in vivo.

BACKGROUND/AIM: Chemotherapeutic approaches involving microtubule-directed agents such as the vinca alkaloids and taxanes are used extensively and effectively in clinical cancer therapy. There is abundant evidence of critical cytoskeletal differences involving microfilaments between normal and neoplastic cells, and a variety of natural products and semi-synthetic derivatives are available to exploit these differences in vitro. In spite of the availability of such potential anti-neoplastic agents, there has yet to be an effective microfilament-directed agent approved for clinical use. Cytochalasins are mycogenic toxins derived from a variety of fungal sources that have shown promising in vitro efficacy in disrupting microfilaments and producing remarkable cell enlargement and multi-nucleation in cancer cells without producing enlargement and multi-nucleation in normal blood cells. Jasplakinolide is a sponge toxin that stabilizes and rigidifies microfilaments. Insufficient in vivo data has been acquired to determine whether any of the microfilament-directed agents have valuable preferential anticancer activity in pre-clinical tumor model systems. This is partly because the limited availability of these agents precludes their initial use in large-scale mammalian pre-clinical studies. Therefore, the present study sought to determine the tolerated in vivo doses of cytochalasins and jasplakinolide in zebrafish (Danio rerio), a well-studied fish cancer model that is 1.5% the size of mice. We also determined the tolerated levels of a variety of clinically active anti-neoplastic agents in zebrafish for comparison with tolerated murine doses as a means to allow comparison of toxicities in zebrafish expressed as µM concentrations with toxicities in mice expressed in mg/kg. MATERIALS AND METHODS: Tolerated doses in zebrafish with various cytochalasins or jasplakinolide were determined by adding the solubilized test agent to water in which the fish were maintained for 24 h, then restored to their normal tanks and monitored for a total of 96 h. RESULTS: Cytochalasin D at 0.2 µM gave an approximate LD50 in zebrafish, while cytochalasin B was fully-tolerated at 5 µM, and gave an LD50 of 10 µM. 21,22-dihydrocytochalasin B was fully-tolerated at 10 µM. Cytochalasin C was tolerated fully at 1 µM, ten-fold higher than the level for cytochalasin D that was tolerated. Jasplakinolide at 0.5 µM did not exhibit any apparent acute toxicity or affect fish behavior for four days, but delayed toxicity was evident at days 4 and 6 when the fish died. Further, the addition of 5 µM glutathione (GSH) at the time of treatment substantially decreased the toxicity of 10 µM cytochalasin B, a level of cytochalasin B that not otherwise tolerated in vivo. Such observations were likely due to GSH-mediated alkylation of C-20 in cytochalasin B, thereby reducing the rate of oxidation to the highly toxic congener, cytochalasin A, and reacting with any cytochalazin A formed. The protective effects of GSH are further supported by its ability to react with α, ß-unsaturated ketone moieties, as is found in cytochalasin A. GSH at 0.8 uM was able to reduce the toxicity of 0.8 µM cytochalasin D, but it took 20 µM GSH to fully protect against the toxicity of 0.8 µM cytochalasin D. CONCLUSION: Pre-clinical evaluation of rare natural products such as microfilamented-directed agents for efficacy in vivo in tumor-bearing zebrafish is a feasible prospect. Dose-limiting toxicities in zebrafish expressed as µM concentrations in water can be used to estimate in vivo toxicities in mice expressed as mg/kg.

The real deal: using cytochalasin B in sonodynamic therapy to preferentially damage leukemia cells.

BACKGROUND/AIM: Sonodynamic therapy (SDT) is a form of ultrasound therapy in which chemotherapeutic agents known as sonosensitizers are administered to increase the efficacy of ultrasound's preferential damage to neoplastic cells. Perhaps one of the most intriguing capabilities of ultrasound is its ability to preferentially lyse cells based on size. Cytochalasin B is a cytokinesis inhibitor that preferentially enlarges and multinucleates malignant cells, making them much more sensitive to ultrasonic irradiation. MATERIALS AND METHODS: The present study investigated the extent of preferential damage inflicted by cytochalasin B on U937 leukemia/human blood cell populations. Cell mixtures were treated with cytochalasin B and then sonicated under a relatively low intensity (3W/cm(2)). RESULTS: Cytochalasin B preferentially damages U937 cells both before and after sonication. This agent also reduces rapid proliferation as the clonogenicity of U937 cells was considerably reduced following treatment. CONCLUSION: Cytochalasin B may have profound therapeutic applications when combined with SDT.