Low Intensity Ultrasound as an Antidote to Taxane/Paclitaxel-induced Cytotoxicity.

The taxane family of compounds, including Taxol/paclitaxel and Taxotere/docetaxel, are surprisingly successful drugs used in combination or alone for the treatment of most major solid tumors, especially metastatic cancer. The drugs are commonly used in regimen with other agents (often platinum drugs) as frontline treatment, or used as a single agent in a dose dense regimen for recurrent cancer. The major side effects of taxanes are peripheral neuropathy, alopecia, and neutropenia, which are grave burden for patients and limit the full potential of the taxane drugs. Especially in the current treatment protocol for peripheral neuropathy, taxane dosage is reduced once the symptoms present, resulting in the loss of full or optimal cancer killing activity. Substantial efforts have been made to address the problem of cytotoxic side effects of taxanes, though strategies remain very limited. Following administration of the taxane compound by infusion, taxane binds to cellular microtubules and is sequestered within the cells for several days. Taxane stabilizes and interferes with microtubule function, leading to ultimate death of cancer cells, but also damages hair follicles, peripheral neurons, and hemopoietic stem cells. Currently, cryo-treatment is practiced to limit exposure and side effects of the drug during infusion, though the effectiveness is uncertain or limited. A recent laboratory finding may provide a new strategy to counter taxane cytotoxicity, that a brief exposure to low density ultrasound waves was sufficient to eliminate paclitaxel cytotoxicity cells in culture by transiently breaking microtubule filaments, which were then relocated to lysosomes for disposal. Thus, ultrasonic force to break rigid microtubules is an effective solution to counter taxane cytotoxicity. The discovery and concept of low intensity ultrasound as an antidote may have the potential to provide a practical strategy to counter paclitaxel-induced peripheral neuropathy and alopecia that resulted from chemotherapy. Taxanes are a class of important drugs used in chemotherapy to treat several major cancers. This article reviews a new laboratory discovery that ultrasound can be used as an antidote for the peripheral cytotoxicity of taxane drugs and discusses the potential development and application of low intensity ultrasound to prevent side effects in chemotherapeutic treatment of cancer patients.

Exposure to low intensity ultrasound removes paclitaxel cytotoxicity in breast and ovarian cancer cells.

BACKGROUND: Paclitaxel (Taxol) is a microtubule-stabilizing drug used to treat several solid tumors, including ovarian, breast, non-small cell lung, and pancreatic cancers. The current treatment of ovarian cancer is chemotherapy using paclitaxel in combination with carboplatin as a frontline agent, and paclitaxel is also used in salvage treatment as a second line drug with a dose intensive regimen following recurrence. More recently, a dose dense approach for paclitaxel has been used to treat metastatic breast cancer with success. Paclitaxel binds to beta tubulin with high affinity and stabilizes microtubule bundles. As a consequence of targeting microtubules, paclitaxel kills cancer cells through inhibition of mitosis, causing mitotic catastrophes, and by additional, not yet well defined non-mitotic mechanism(s). RESULTS: In exploring methods to modulate activity of paclitaxel in causing cancer cell death, we unexpectedly found that a brief exposure of paclitaxel-treated cells in culture to low intensity ultrasound waves prevented the paclitaxel-induced cytotoxicity and death of the cancer cells. The treatment with ultrasound shock waves was found to transiently disrupt the microtubule cytoskeleton and to eliminate paclitaxel-induced rigid microtubule bundles. When cellular microtubules were labelled with a fluorescent paclitaxel analog, exposure to ultrasound waves led to the disassembly of the labeled microtubules and localization of the signals to perinuclear compartments, which were determined to be lysosomes. CONCLUSIONS: We suggest that ultrasound disrupts the paclitaxel-induced rigid microtubule cytoskeleton, generating paclitaxel bound fragments that undergo degradation. A new microtubule network forms from tubulins that are not bound by paclitaxel. Hence, ultrasound shock waves are able to abolish paclitaxel impact on microtubules. Thus, our results demonstrate that a brief exposure to low intensity ultrasound can reduce and/or eliminate cytotoxicity associated with paclitaxel treatment of cancer cells in cultures.

Nuclear Lamin A/C Expression Is a Key Determinant of Paclitaxel Sensitivity.

Paclitaxel is a key member of the Taxane (paclitaxel [originally named taxol], docetaxel/Taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anticancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from lamin A/C-null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wild-type cells. Forced overexpression of lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved by either the withdrawal of serum or the addition of mitotic inhibitors. These results provide a new understanding for the mitotis-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear lamin A/C protein levels correlate with nuclear shape deformation and are a key determinant of paclitaxel sensitivity of cancer cells.

Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation.

Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.

Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles.

Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis) or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila). The bacteria described in this review often use secretion systems to control the host's response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII) are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

Autophagy and proteins involved in vesicular trafficking.

Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway.

The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.