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.

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.