SummaryBackground 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.

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The hybrid lactic dehydrogenases H2M2 from chicken liver and HMS from chicken leg muscle have been isolated and crystallized. The pure H4 form of the enzyme has been isolated and crystallized from chicken liver and compared to the H4 obtained from heart. Fingerprint patterns of tryptic digests of the hybrids have been compared to patterns obtained from a 1:1 mixture of the H4 and M4 and have shown the hybrids to be a combination of the pure forms. Amino acid analyses, particularly of histidine, place the hybrids at points intermediate between the pure types. The H4 from chicken liver did not vary significantly in amino acid content or molecular weight from the H4 obtained from chicken heart. Molecular weights of the hybrids were in the same range as those of the pure types. Heat and time stability studies of the chicken hybrids and of beef hybrids isolated from starch grain have established the intermediate nature of the hybrids. Immunological properties, analog ratios, and oxalate inhibition also demonstrate that the hybrids are combinations of the H4 and M4 forms of the enzyme.The evidence set forth on the developmental characteristics of lactic dehydrogenase in chicken (Cahn et al., 1962) and in mammals has indicated that the lactic dehydrogenases of these animals are composed of two types of subunits which may be combined to yield five electrophoretically distinguishable enzymes. The lactic dehydrogenase of beef heart has been dissociated in the presence of 5 m guanidine into subunits of approximately 35,000 mw (Appella and Markert, 1961). The beef heart and chicken heart enzymes similarly have been dissociated with sodium dodecylsulfate (Di Sabato and Kaplan, 1964). The intermediate lactic dehydrogenases have recently been made in vitro by incubation of the extreme electrophoretic types (Markert, 1963).In this paper we shall refer to the subunits of lactic dehydrogenase either as H or M type and to the native enzymes as H4, H3M, H2M2, HMS, and M4 in order of their decreasing mobility toward the anode at pH 7.0. This paper is concerned with the isolation and characterization of two of the hybrid lactic dehydrogenases, HMj and H2M2, from chicken tissues.These characterizations are compared with the results for the M4 and H4 types. Analysis of the amino acid compositions and "fingerprint" patterns of these enzymes yielded results consistent with the hypothesis that these enzymes consist of two distinct polypeptide subunits.1 Per cent saturation was based on Table I in S. P. Colo wick and N. O. Kaplan (eds.). Methods in Enzymology, Vol. I., New York, Academic Press (1955), p. 76, even though the enzymatic solutions were kept at 4°.

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.

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.