Bioelectronics of cellular cytoskeleton; Monitoring the conducting

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Bioelectronics of cellular cytoskeleton; Monitoring the conducting variation of cytoskeleton to sensing the drug resistance Milad Gharooni, Alireza Alikhani, hassan moghtaderi, Hamed Abiri, Alireza Mashaghi, fereshteh Abbasvandi, mohammad ali khayamian, zohreh sadat miripour, Ashkan Zandi, and Mohammad Abdolahad ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01142 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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Bioelectronics of cellular cytoskeleton; Monitoring the conducting variation of cytoskeleton to sensing the drug resistance Milad Gharooni a,b,#, Alireza Alikhani a,b,#, Hassan Moghtaderia,b, Hamed Abiri a,b, Alireza Mashaghi c, Fereshteh Abbasvandi d , Mohammad Ali Khayamian a,b, Zohreh sadat Miripour a,b, Ashkan Zandi a,b, ,Mohammad Abdolahad a,b,*,# a Nano

Electronic Center of Excellence, Nano Bio Electronic Devices Lab, School of Electrical and

Computer Engineering, University of Tehran, Tehran, Iran, P.O. Box 14395/515 b Nano

Electronic Center of Excellence, Thin Film and Nanoelectronic Lab, School of Electrical and

Computer Engineering, University of Tehran, Tehran, Iran, P.O. Box 14395/515 c Leiden

Academic Centre for Drug Research, Faculty of Mathematics and Natural Sciences, Leiden

University, Leiden, The Netherlands. d

Department of Surgery, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, P. O. Box

15179/64311 Tehran, Iran. # Equally contributing authors

* Corresponding Author:

[email protected]

ABSTRACT Actin and microtubules form cellular cytoskeletal network which mediates cell shape, motility and proliferation, and are key targets for cancer therapy. Changes in cytoskeletal organization dramatically affect mechanical properties of the cells and correlate with proliferative capacity and invasiveness of cancer cells. Changes in cytoskeletal network expectedly leads to altered nonmechanical material properties including electrical conductivity as well. Here we applied for the first time microtubule and actin based electrical measurement to monitor changes in the electrical properties of breast cancer cells upon administration of anti-tubulin and anti-actin drugs respectively. Semi-conductive behavior of microtubules and conductive behavior of actins

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presented different bioelectrical responses (in similar frequencies) of the cells treated by antitubulin with respect to anti actin drugs. Doped silicon nanowires were applied as the electrodes due to their enhanced interactive surface and compatibility with electronic fabrication process. We found that treatment with Mebendazole (MBZ), a microtubule destabilizing agent, decreases electrical resistance while treatment with Paclitaxel (PTX), a microtubule stabilizing agent, leads to an increase in electrical resistance. In contrast, actin destabilizing agents, Cytochalasin D (CytD) and actin stabilizing agent, Phalloidin, lead to an increased and decreased electrical resistance respectively. Our study thus provides proof-of-principle of the usage of determining the electrical function of cytoskeletal compartments in grading of cancer as well as drug resistance assays. KEYWORDS: Cytoskeleton, Cancer cell, Impedance, Label-free method, Drug response. Microtubules (MTs) and actin are dynamic filamentous cytoskeletal proteins1–3, and have been identified as important therapeutic target in cancer cells. MTs are constantly lengthening and shortening throughout the cell cycle 4 and are involved in many crucial cellular functions such as generating cell movement

5,

intracellular trafficking

6,

and intracellular macromolecular

assemblies (e.g., dynein and kinesin) 7. Cellular actin network is also highly dynamic structure and can remodel into a variety of architectures including branched or cross-linked networks, parallel bundles, and antiparallel structures8. Organization of microtubules and actin network are critically important for cancerous transformation, invasion and metastasis9,10. Importantly, alterations in MT stability and modification as well as the expression of different tubulin isotypes and microtubule-associated proteins (MAPs) have been demonstrated for a range of cancers including colon and breast

11,12.

These changes have been correlated with poor prognosis and

chemotherapy resistance in solid and hematological cancers11. Intense research is underway to develop microtubule-binding agents with enhanced tumor specificity, reduced toxicity and enhanced insensitivity to resistance mechanisms13,14. Actin remodeling agents are also being considered as anti-cancer drug options15. Various technologies have been developed and used to monitor the effect of anti-cancer drugs such as microtubule or actin remodeling agents. These techniques typically measure cell growth or 2 ACS Paragon Plus Environment

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proliferation. Various assays have been developed to assess the effects of drugs on cancer cell growth and proliferation such as adenosine 5´-triphoste (ATP)16, the fluorescent cytoprint (FCP)17, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)18,19, the differential staining cytotoxicity (DiSC)20, 3H-Uridine, the extreme drug resistance (EDR)21, flow cytometry22, immunoblotting 23 and immunofluorescence confocal assays24,25. In the recent years, efforts have been applied to develop new assays for label-free and simple analysis while maintaining the accuracy26. Technologies that directly inform on organization of microtubule or actin would also be helpful in measuring anti-cancer drug effect or drug resistance. Microtubule and actin filaments are active materials with respective mechanical, optical and electrical properties that could be used to inform on their structure. Mechanical analyses of cancer cells have been widely performed using various techniques including force spectroscopy. Electrical properties of these networks could in principle be used to inform on their structure in vivo. In vitro analysis of MTs and actin reveals that they are semi-conductive27,28 and conductive29,30,31 respectively. However, there is currently no report where conductivity measurements have been used to study cytoskeletal organizations within cancer cells. In this article, we develop and use a nanowire-based sensing platform to measure the electrical resistance of cancer cells. We choose to work on breast cancer and correlate the electrical resistance of different untreated breast cancer cells with their invasiveness grade (MCF-7, MDAMB-231, MDA-MB-468 and MCF-10A)

32–34

and compare the electrical resistance of untreated

cancer cells with those treated by cytoskeletal drugs. We apply the proposed approach for fast detection of Mebendazole (MBZ) and paclitaxel (PTX)35, two widely investigated anti-tubulin drugs36,37, and Cytochalasin D (CytD) and Phalloidin, two widely investigated anti-actin drugs, on breast cancer cells. Silicon nanowires as new generation of electrodes in bioelectrical 3 ACS Paragon Plus Environment

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measurements, 38–43 are applied to enhance the interactive surface by the attached cells4435. In this approach, as we demonstrate, reliable resistance of the cells could be measured even after their treatment with low doses of drugs. We confirm our results by confocal and fluorescent microscopy images and compare them with flow cytometry, Measurement of Intracellular ROS, Measurement of the mitochondrial membrane potential (MMP), RT-PCR analysis, Nitrite (NO2-) detection and Annexin/V PI (API) assays to evaluate the accuracy of the introduced method. Furthermore, we use the technology to study the effect of actin remodeling agents and compare the electrical properties of cells treated with MT agents with those treated with actin remodelers. Finally, we develop a simple theoretical framework to explain the observations.

Results and Discussion We start by hypothesizing that reorganization of MTs and actin network affects the conductivity of cells (Figure 1). The lengthening and distribution of the cytoskeletal filaments (Figure 1A) might change the impedance response due to the penetration of current lines into the cells interior (Figure 1B). We aim to match the mean impedance response diagram of the cancer cells by the effect of anti-tubulin drugs on their function and metabolism. We hypothesize that enhanced density of the MTs because of polymerization (Figure 1A2) reduce the passed current and increase the impedance responses (Figure 1B2). In contrast, reduced number of MTs because of depolymerization (Figure 1A3) must increase the passed currents through the cells and subsequently, decrease the mean impedance of the cells (Figure 1B3). The effect of actin polymerization/depolymerization is expected to be completely reverse. Because actin filaments are conductive components of cytoskeletal structure, polymerization and de-polymerization of actins enhanced (Figure 1C1) and decreased (Figure 1C2) the current density passed through the cells respectively.


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Figure 1. (A1 to A3) schematic images of extensive de-polymerization and polymerization rate in MTs resulted in non-regular spindle formation in MBZ (10.5nM) and PTX (1nM) treated samples compared to the control sample, respectively. (B1 to B3) schematic of current blocking by MTs with decreasing current for higher polymerization of MTs. (C1 to C2) schematic of current penetration into the cell with polymerized (C-1) and depolymerized (C-2) actins in presence of CytoD (1µM) and Phalloidin (10uM). Actins and MTs have reverse effect on the electrical resistance of the cells due to their polymerization/depolymerization.

To test our hypothesis, we fabricated silicon nanowire (SiNW) microelectrodes to enhance the interactive surface between incoming currents and investigated cells. The crystallinity and composition of SiNWs, which directly impact their role as electrical electrodes, were analyzed by Raman spectroscopy, X-ray diffraction (XRD), selected area diffraction (SAED) and Energydispersive X-ray (EDX) spectroscopy all presented in supplementary (Section S2; Figure S1).


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Figure 2A presents SEM images of the SiNW array in the architecture of biosensor with interdigital transducers (IDTs). Such decoration of silicon nanowire instead of the Au electrode would increase the interactive surface and impedance sensitivity of the sensor45.

We investigated the

biocompatibility of the fabricated SiNW by MTT assays (Figure 2B) and found 95 and 90% viability for the seeded MCF-7 cells on un-doped and doped SiNWs after 24h in comparison with control sample respectively. This revealed the maintenance of interactive contact sites between NWs and the MCF-7 in all states of proliferation as depicted in supplementary (Section S2; Figure S2). Florescent microscopy images from Acridine Orange (AO) stained cancer cells attached on SiNW arrays (Figure 2C) indicated the vital state of the cells after long time interaction with the wires as expression of bright yellow color in stained cells would be correlated with their live state4647. Shape and geometry of cancer cells attached on SiNWs are observable in Figure 2D and 2E. The distribution of nanowires (NWs) induced large efficient 3D interactive surface in interaction with the cells observed in FESEM (Figure 2D) and AFM images (Figure 2E). To further clarify the response of SiNWs as the electrodes of ECIS, we compared the results of impedance determined by SiNW-ECIS with those measured by Au-ECIS in supplementary (Section S2; Figure S3). The enhanced interactive surface between SiNWs and the cells’ membrane play the key role in improved signals extracted by SiNW-ECIS. Increased impedance of control cancer cells during growth and proliferation would be inferred from the supplementary (Section S2; Figure S4).


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Figure 2. (A): SEM image of SiNW electrodes, (B): The Biocompatibility results of SiNW investigated by MTT assays and (C): Florescent microscopy images of cancer cells attached on SiNW arrays, (D): SEM images of cancer cells attached on the SiNW, (E): AFM image of cancer cells attached on the SiNW.

Before investigating the electrical characteristics of drug treated cells, biological effects of MT (PTX and MBZ) and actin (CytD and Phalloidin) targeted drugs were assayed by wide variety of tests such as NO, ANVPI, Cell cycle, RT-PCR, MMP, ROS as discussed in supplementary (Section S3; Figures S5-S8; Tables S1-S3). The concentrations of PTX, MBZ, CytD and Phalloidin drugs were selected to be 1Nm48, 10.5Nm49503551, 1µm52 and 10um53 respectively. Increased and Decreased impedance in MT polymerized and depolymerized cells Figure 3 shows the comparative impedance presentation of low and high grades of breast cancer (MCF- 7, MDA-MB-231, and MDA-MB-468) and healthy cells (MCF-10A) in subsequent intervals of time. The impedance changes were measured with respect to the impedance of the sensor in 4 hours after dropping the cells on the SiNW electrodes (this time would be the reference time in which the cells were seeded on the SiNW arrays). In this regards the time intervals were defined as T=0, 5, 8, 20 which referred to 4, 9, 12 and 24 hours after dropping the cells on the electrodes. 7 ACS Paragon Plus Environment

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Figure 3. (A1 to D1) and (A2 to D2) average impedance and phase of MCF-10A, MCF-7, MDA-MB231 and MDA-MB-468 cells for T=0 to T=20, respectively. (A3 to D3) and (A4 to D4) average impedance and phase of MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by 1nM PTX for T=5 to T=20, respectively. (A5 to D5) and (A6 to D6) average impedance and phase of MCF-10A, MCF-7, MDA-


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MB-231 and MDA-MB-468 cells treated by 10.5nM MBZ for T=5 to T=20 respectively. Also, T=5, T=8 and T=20 refer to 5, 8, 20 hours after attachment of cells.

Such increment for control samples (Figure 3A1-3D1 and 3A2-3D2 from T=0 to T=20) is in a good agreement with enhanced index of attached cells54. Dielectric properties of the cytoskeleton resulted in current blocking and impedance increment based on Beta and Gamma dispersions as the intrinsic bioelectrical properties of the cell in response to AC electrical excitation in higher frequencies

55.

Equivalent circuit model was also discussed in supplementary. The interactive

surface between the cells and electrodes would be crucial in the quality of extracted signals. The increase in the impedance (decrease in the phase) response of the sensor is the obvious result of the increased cell index (Figure 3A1-3D1 and 3A2-3D2 from T=0 to T=20). As seen in this figure, as cell get more cancerous (MCF-10A vs MCF-7) or metastatic (MCF-7 to MDA-MB-231 and MDA-MB-468) ability of the cell to block the passing current due to beta dispersion decreased due to degradation of the membrane integrity and changed ratio between phospholipids and fatty acids as well as lipoproteins and thus impedances is lower for the same number of the cells at first hours. But mitotic rate of the cancerous and metastatic cells are more than healthy cells and therefore the rate of impedance increment (phase increment in negative regime) due to enhanced coverage rate of electrodes by the proliferated cells are faster. Figure 3 (A3 to D3) and (A4 to D4) show the electrical response of PTX treated healthy and cancer cells in similar time lapses. Mean impedance responses of the PTX (1nM) treated cells in high frequencies followed an additive regime. Our proposed mechanism is directly related to the electrical properties of polymerized MTs after PTX treatment. As we mentioned, MTs are protein filaments with semiconducting behavior (90 ohm-1.m-1)56. The over polymerized MTs (because of PTX treatment) would be a dense dielectric media which block the flowing current into the cells and increase the impedance of the sensor with respect to non-treated cells (Figure 3A1 to 3D1). 9 ACS Paragon Plus Environment

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The phase responses of PTX treated cells (Figure 3A4 to 3D4) corroborated the impedance responses. Increment in impedance due to MT polymerization is equal to increment of the phase absolute values. In MDA-MB-231 and MDA-MB-468 (as metastatic cancer cell lines), impedance diagrams presented the MT polymerization by increase from T=5 to T=20 (Figure 3A3 to 3D3). In addition, the mean impedance values exhibited decrease in negative regime as a corroboration for increased dielectric media in cytoplasm (could be observed by capacitive behavior) (Figure 3A4 to 3D4). By comparison of impedances for PTX treated sample (figure 3A3 to 3D3 and 3A4 to 3D4) by control samples (Figure 3A1 to 3D1 and 3A2 to 3D2) it can be inferred that for PTX treated samples mean impedance didn’t change significantly in first hours and just a little decrease respect to the control sample can be seen but this difference gets more in as time going. But the phase of the PTX treated samples have their increment in negative regime like control samples. Microtubules of PTX treated samples have been polymerized which results in more capacitive behavior (more negative phase) of the cells. But at the same time PTX treated samples arrested in mitotic function and more apoptosis accrued for drug treated samples and thus mean impedance didn’t increase as fast as control samples. In addition, metastatic cells (MDA-MB-231 and MDAMB-468) are more resistive to the drug treatment and impedance didn’t change as more as nonmetastatic (MCF-7) cells. Moreover, the MCF-10A cells didn’t change significantly by PTX treatment. In contrast to PTX, treating the cells with MBZ (depolymerizing agents) observably reduced the impedance of the cells which could be explained by our proposed mechanism based on MT depolymerization. We know that the overall conductance of the cytoplasm is 2 orders of magnitude higher than the MTs 56. When the MTs being depolymerized by the MBZ, the ratio of dielectric media in the cytoplasm would be reduced in comparison with conductive region. Hence, high 10 ACS Paragon Plus Environment

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frequency current penetration into the cells would be increased and impedance of the MBZ treated cells would be decreased all of which was observed in the responses of the sensor (Figure 3A5 to 3D5). Moreover, impedance phase values decreased in negative regime which supports the degradation of dielectric components of the cytoplasm (Figure 3A6 to 3D6). The current penetration would be enhanced from the MT free cytoplasmic regions as detected by impedance reduction in the response of the biosensor. To observe the depolymerizing effect of MBZ on MDAMB231 and MDA-MB-468 cells we ought to increase the dose of the drug to 10.5nM. High frequency impedance and phase responses showed the sharp decrease caused by MT depolymerization. In addition, metastatic cells (MDA-MB-231 and MDA-MB-468) are more immune to the drug treatment and impedance didn’t change as more as non-metastatic (MCF-7) cells.

Figure 4. A-1 to A-2 show the corresponding confocal images of MCF-7 non-treated cells (control), PTX (B-1 and B-2) and MBZ (C-1 to C-2) MCF-7 treated samples after 48h drug incubation.

If we assume the MT distribution presented in 4A-1 and 4A-2 as the reference image taken from control MCF-7 cell, MT polymerization and de-polymerization in the drug treated cells could be


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well understood. Confocal imaging from the PTX treated cells showed increased aggregation in MT polymers (Figure 4B-1 and 4B-2) in comparison with control cell (Figure 4A-1 and 4A-2). Such additive dielectric component increased the electrical responses of PTX treated cancer cells. Confocal images taken from the MBZ treated cells showed the MT de-polymerization in which the monstered shapes of the MTs are observable (Figure 4C-1 and 4C-2). Reduced impedance of MBZ treated cells could be attributed to the degraded dielectric components (MTs) in the cytosol in the first 8hr after treatment.

Impedance response of actin polymerized/depolymerized cells Next, we studied the contributions of actin network to electrical responses measured by our sensors. For this aim, we used actin remodeling drugs and recorded the electrical response before and after treatment. The electrical resistance of the cancer cells treated by Cytochalasin D and Phalloidin as actin polymerizer and de-polymerizer agents were determined respectively. Figure 5 presents the data from MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by Cytochalasin D (inducing cell death by suppressing actin polymerization)57. Our data reveals that a reduced concentration of actins (Figure 5 A1 to D1) results in an increased resistance of the Cytochalasin D (CytD) treated cells from all phenotypes. Increased phase values in negative regimes corroborated the impedance results of CytD treated cells (Figure 5A2-D2). The phase of the CytD treated samples have their increment in negative regime like control samples. Mechanism of the CytD effect on cells can be deduced from this comparison, CytD treated samples have depolymerized actins which is result in more capacitive behavior (more negative phase) of the cells. In contrast, Phalloidin (as an actin polymerizing agent)

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treated cells exhibit decreased

electrical resistance (Figure 5 A3 to D3) which can be attributed to an increased concentration of actins, as a conductive component of the cytoskeleton, consistent with due to polymerizing effect 12 ACS Paragon Plus Environment

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of Phalloidin (10uM). Decreased values of impedance phase in negative regimes indicated the reduced impedance of the Phalloidin-treated cells in all of the lines (Figure 5 A4 to D4). These results corroborated that the role of actin disassembly in total resistance of the cancer cells is in contrast to MTs. In addition, metastatic cells (MDA-MB-231 and MDA-MB-468) are more immune to the drug treatment and impedance didn’t change as more as non-metastatic (MCF-7) cells.

Figure 5. (A1 to D1) and (A2 to D2) average impedance and phase of MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by 1μM CytD for T=5 to T=20, respectively. (A3 to D3) and (A4 to D4) average impedance and phase of MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by Phalloidin for T=5 to T=20, respectively. . Also, T=5, T=8 and T=20 refer to 9, 12, 24 hours after cells dropping on the sensors. Note: T=5, T=8 and T=20 refer to 5, 8 and 20 hours after attachment of the cells.


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Confocal images of the breast cancer cells treated by CytD showed the disassembly of the actins with respect to control samples in all of the cell lines (Figure 6 A2-B2-C2). Moreover, confocal images of Phalloidin treated cells presented to over polymerized deregulation in actins in correlation with increased conductance (decreased resistance) of the cytoplasm (Figure 6 A3-B3C3).

Figure 6. A-1 to C-1 show the corresponding confocal images of non-treated cells (control), Cytochalasin D (A-2 to C-2) and Phalloidin (A-3 to C-3) treated MCF-7, MDA-MB-231 and MDA-MB-468 samples after 48h drug incubation.

Table 1: Impedance and phase behavior of the different treated samples & effects of the drugs on cytoskeletal structures.


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Phalloidin

CytD

PTX

MBZ

Control

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|Impedance|

↑↑

↓↓

↑

↑

↓↓

|phase|

↑↑

↓↓

↑↑

↑↑

↓↓

Effect on Microtubules

-

Over

Over

-

-

De-polymerization

polymerization

-

-

Over

Over

de-polymerization

polymerization

Effect on Actins

-

Table 1 summarized the impedance and absolute phase behaviors of different treated samples. Control samples showed time dependent increase in their electrical parameters meanwhile MBZ and Phalloidin treated samples showed decreasing electrical characteristic regime.

Figure 7. Normalized impedance of the MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by MBZ, PTX, Phalloidin and CytD. Note: T=5, T=8 and T=20 refer to 5, 8 and 20 hours after attachment of the cells.


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In the case of PTX and CytD treated samples, impedance increased slowly for short time after treatment but their phase increased fast due to polymerized tubulin and depolymerized actin which were resulted in a more capacitive behavior of the cells. Figure 7 shows normalized impedance of the MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by MBZ, PTX, Phalloidin and CytD. Impedances normalized to the impedance of cell in T=0. As seen in figure 7A and 7 B, normalized impedance and absolute phase of MBZ treated cells showed time evaluated decreasing regime. In PTX treated samples, these parameters were ascending but not as much as control samples. As mentioned before such behavior in PTX treated cells is due to over-polymerized tubules. Phalloidin treated samples showed descending behavior in their electrical parameters (figure 7C) such as observed in MBZ treated samples. CytD treated samples exhibited an ascending electrical parameters (figure 7D) such as observed in PTX treated samples. Electrical Resistance of drug treated lysed cells To further confirm the observed conductivity changes after actin or tubulin polymerization/depolymerization, we measured the conductivity of the cytoplasmic solution by lysing the control and drug treated cells followed by determining their resistivity (ρ) (Figure 8). The control and treated cells were lysed due to the procedures mentioned in experimental section. As the main part of the lysed solution of the cells is cytosol, the electrical characterization of this aqua would exhibit a strong correlation with electrical parameters of the cytoplasm. Figure 8, presents the resistivity profiles of the cytosol in all types of the cancer cells treated by actin and microtubule remodeling drugs. It can be observed that the ρ of the PTX treated cells was increased, while it was decreased for cells treated with MBZ. The data revealed that the over polymerized/depolymerized MTs increased/decreased the resistivity of cytoplasm. In addition, over-polymerized actins in the cytosol of Phalloidin treated cells reduced the resistivity while this


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value was increased in cytosols of depolymerized actins derived from CytD treated cells. Dielectric and conductive characteristics of MTs and actins resulted in their reverse effect on electrical parameters of cytoplasm respectively (Figure 8).

Figure 8. (A-D) resistivity of the lysed MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cells treated by MBZ, PTX, Phalloidin and CytD.

Conclusion In this study, we measured the contributions of microtubule and actin to the electrical properties of cells. We performed this study by analyzing cancer cells that have been subjected to actin or microtubule remodeling drugs and demonstrate that electrical properties inform on cancer cell invasiveness and drug resistance. Doped silicon nanowires were applied as electrodes to enhance the interactive surface during the measurement. Confocal, florescent, API and flow cytometry assays were applied to elaborate the structure and function of drug induced cells and evaluate the trends of cells’ resistance with respect to physiological states. Our data reveals that remodeling of actin and microtubule networks affect the electrical properties in opposite ways, in agreement with the reported electrical properties of individual filaments in vitro. We confirmed our in measurements with in vitro measurements of lysed cells. Finally, our proposed equivalent circuit model explains the impact of the cells’ electrical resistance (induced by the anti-cytoskeletal 17 ACS Paragon Plus Environment

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drugs) in the total impedance of the sensor, in agreement with our experimental data. Functional correlation between MT or actin disassembly and resistive responses of the cells was observed in all phenotypes of breast cells ranged from normal to metastatic grades. Our study demonstrate that the technology used here could be used as a label-free method for monitoring the extreme drug resistance (EDR) of cancer cells.

Materials and methods Fabrication of SiNW electrodes Silicon substrate was cleaned through standard RCA #1 cleaning method (NH4OH:H2O2:H2O solution and volume ratio of 1:1:5 respectively). In order to insulate the substrate, SiO2 layer (~250 nm) was grown on the substrate by wet oxidation furnace59. Then, a thin layer of gold (7-9 nm) was coated on the oxide as catalyst layer for growth of SiNWs. The deposition was carried by sputtering system (Veeco Co.) begun at a base pressure of 10−6 Torr. Subsequently, the gold layer was patterned by a standard optical lithography process and wet etching of Au to form the electrodes. The growth of nanowires was achieved in a LPCVD system (SensIran Co.) by the assistance 30-40 SCCM SiH4 gas and a total pressure of 0.5 Torr and 500°C for 10min. The SiNW was grown on patterned Au layer with ECIS pattern. Cell culture MCF-10A, MCF-7, MDA-MB-231 and MDA-MB-468 cell lines, obtained from the National Cell Bank of Iran (Pasteur Institute). Cells were maintained at CO2 incubator (37 °C, 5% CO2) in RPMI1640 medium (Sigma) supplemented with 5% fetal bovine serum (Sigma), and 1% penicillin/ streptomycin (Gibco). The fresh medium was replaced every other day. Prior to each experiment, cells were trypsinized to be detached from the substrate and resuspended on the SiNW surface. To minimize the effect of trypsinization, the procedure was taken less than 4 minutes at room


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temperature around 20-22oC 60.We held the samples in incubator for 4h to achieve cells attachment on the SiNWs and the floating cells washed off. Then the MBZ, PTX, CytD and Phalloidin drugs concentrations were added to individual samples. The signal recording and biological assays were investigated 5, 8 and 20 h after the drug treatment (9, 12 and 24h after the beginning of culturing process respectively). Impedance measurement Measurements were performed with applied voltage of 400 mV. The real-time monitoring of the cells activities was performed by measuring the impedance at frequencies ranging from 400 Hz to 40 kHz in the time interval between 4h to 24h after the cells were dropped on the SiNWs. The best response of the cells membrane to stimulating signals were reported in such range of frequencies 55,61,62.

To be ensure from the impedance response, we repeated the electrical measurements 10

times in five different frequencies (ranged between 400-40000Hz) for each sensor. Cell lysing method First, the cells were detached from the wells by trypsinization, subsequently they were centrifuged (1200rpm, 3min)63. Then we removed the solution and added PBS to the falcone contained the cells. The falcon was centrifuged (2000rpm for 20sec) to remove the PBS and then we added Lysis-buffer (cell lysis NP40 buffer CMG) to the Falcone (60µl of the Lysis buffer to each 106 cells)64. Confocal imaging Cells were grown on individual glass slides and treated with MBZ, PTX, CytD and Phalloidin for 24h. Also control sample was prepared as reference for comparison. Samples were then washed with PBS and permeabilized with MTs stabilizing buffer [80 mM PIPES-KOH (pH 6.8), 5 mM EGTA, and 1 mM MgCl2 containing 0.5% Triton X-100] for 5 minutes at room temperature before 19 ACS Paragon Plus Environment

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being fixed with chilled absolute methanol for 10 min at –20°C. Fixed cells were washed and incubated with monoclonal mouse anti-α-tubulin antibody (Sigma) for 1 hour at room temperature followed by incubation with FITC-conjugated anti-mouse IgG antibody (Santa Cruz Biotechnology). The stained cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and observed by confocal microscopy65. Supporting Information Available: The following files are available free of charge. Support information: Additional electrical and biological analyses, some methods of imaging and fabrication processes

References (1)

Ghosh, S.; Mohapatra, S.; Thomas, A.; Bhunia, D.; Saha, A.; Das, G.; Jana, B.; Ghosh, S. Apoferritin Nanocage Delivers Combination of Microtubule and Nucleus Targeting Anticancer Drugs. ACS Appl. Mater. Interfaces 2016, 8 (45), 30824–30832.

(2)

Jana, B.; Mohapatra, S.; Mondal, P.; Barman, S.; Pradhan, K.; Saha, A.; Ghosh, S. αCyclodextrin Interacts Close to Vinblastine Site of Tubulin and Delivers Curcumin Preferentially to the Tubulin Surface of Cancer Cell. ACS Appl. Mater. Interfaces 2016, 8 (22), 13793–13803.

(3)

Wu, Z.; Nogales, E.; Xing, J. Comparative Studies of Microtubule Mechanics with Two Competing Models Suggest Functional Roles of Alternative Tubulin Lateral Interactions. Biophys. J. 2012, 102 (12), 2687–2696.

(4)

Dumont, S.; Mitchison, T. J. Compression Regulates Mitotic Spindle Length by a Mechanochemical Switch at the Poles. Curr. Biol. 2009, 19 (13), 1086–1095.

(5)

Ananthakrishnan, R.; Ehrlicher, A. The Forces behind Cell Movement. Int. J. Biol. Sci. 2007, 3 (5), 303–317.

(6)

Hammond, J. W.; Cai, D.; Verhey, K. J. Tubulin Modifications and Their Cellular Functions. Curr. Opin. Cell Biol. 2008, 20 (1), 71–76.

(7)

Roberts, A. J.; Kon, T.; Knight, P. J.; Sutoh, K.; Burgess, S. A. Functions and Mechanics of Dynein Motor Proteins. Nat. Rev. Mol. Cell Biol. 2013, 14 (11), 713–726.

(8)

Blanchoin, L.; Boujemaa-Paterski, R.; Sykes, C.; Plastino, J. Actin Dynamics, Architecture, and Mechanics in Cell Motility. Physiol. Rev. 2014, 94 (1), 235–263.

(9)

Bordeleau, F.; Alcoser, T. A.; Reinhart-King, C. A. Physical Biology in Cancer. 5. The 20 ACS Paragon Plus Environment

Page 20 of 26

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

Rocky Road of Metastasis: The Role of Cytoskeletal Mechanics in Cell Migratory Response to 3D Matrix Topography. Am. J. Physiol. Cell Physiol. 2014, 306 (2), C110-20. (10)

SURESH, S. Biomechanics and Biophysics of Cancer Cells☆. Acta Biomater. 2007, 3 (4), 413–438.

(11)

Parker, A. L.; Kavallaris, M.; McCarroll, J. A. Microtubules and Their Role in Cellular Stress in Cancer. Front. Oncol. 2014, 4, 153.

(12)

Orr, G. A.; Verdier-Pinard, P.; McDaid, H.; Horwitz, S. B. Mechanisms of Taxol Resistance Related to Microtubules. Oncogene 2003, 22 (47), 7280–7295.

(13)

Dumontet, C.; Jordan, M. A. Microtubule-Binding Agents: A Dynamic Field of Cancer Therapeutics. Nat. Rev. Drug Discov. 2010, 9 (10), 790–803.

(14)

Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R. Drugs That Target Dynamic Microtubules: A New Molecular Perspective. Med. Res. Rev. 2011, 31 (3), 443–481.

(15)

Rao, J.; Li, N. Microfilament Actin Remodeling as a Potential Target for Cancer Drug Development. Curr. Cancer Drug Targets 2004, 4 (4), 345–354.

(16)

Kimura, Y.; Shibasaki, S.; Morisato, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Matsuo, M.; Amachi, T.; Ueda, M.; Ueda, K. Microanalysis for MDR1 ATPase by HighPerformance Liquid Chromatography with a Titanium Dioxide Column. Anal. Biochem. 2004, 326 (2), 262–266.

(17)

Hoffman, R. M. In Vitro Sensitivity Assays in Cancer: A Review, Analysis, and Prognosis. J. Clin. Lab. Anal. 1991, 5 (2), 133–143.

(18)

Plumb, J. A.; Milroy, R.; Kaye, S. B. Effects of the PH Dependence of 3- ( 4 , 5Dimethylthiazol-2-Yl ) -2 , 5-Diphenyl- Tetrazolium Bromide-Formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-Based Assay1. CANCER Res. 1989, 49(August), 4435–4440.

(19)

Mirakabadi, A. Z.; sarzaeem, A.; Moradhaseli, S.; Sayad, A.; Negahdary, M. Necrotic Effect Versus Apoptotic Nature of Camptothecin in Human Cervical Cancer Cells. Iranian Journal of Cancer Prevention. 2012, 5(3), 109–116.

(20)

Su, Y. Cancer Chemosensitivity Testing : Review. J. Cancer Ther. 2014, 5(7), 672–679.

(21)

d’Amato, T. A.; Pettiford, B. L.; Schuchert, M. J.; Parker, R.; Ricketts, W. A.; Luketich, J. D.; Landreneau, R. J. Survival among Patients with Platinum Resistant, Locally Advanced Non-Small Cell Lung Cancer Treated with Platinum-Based Systemic Therapy. Ann. Surg. Oncol. 2009, 16 (10), 2848–2855.

(22)

Gupta, V.; Zhang, Q.-J.; Liu, Y.-Y. Evaluation of Anticancer Agents Using Flow Cytometry Analysis of Cancer Stem Cells. Methods Mol. Biol. 2011, 716, 179–191.

(23)

Ayyagari, V. N.; Brard, L. Bithionol Inhibits Ovarian Cancer Cell Growth in Vitro Studies on Mechanism(s) of Action. BMC Cancer 2014, 14 (1), 61.

(24)

Lovitt, C. J.; Shelper, T. B.; Avery, V. M. Advanced Cell Culture Techniques for Cancer Drug Discovery. Biology (Basel). 2014, 3(2), 345–367. 21 ACS Paragon Plus Environment

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(25)

Olivo, M.; Lau, W.; Manivasager, V.; Hoon, T. P.; Christopher, C. Fluorescence Confocal Microscopy and Image Analysis of Bladder Cancer Using 5-Aminolevulinic Acid. Int. J. Oncol. 2003, 22 (3), 523–528.

(26)

Alikhani, A.; Gharooni, M.; Moghtaderi, H.; Farokhmanesh, F.; Abiri, H.; Salimi, M.; Attari, F.; Abdolahad, M. An Electrochemical Biosensor to Distinguish between Normal and Cancer Cells Based on Monitoring Their Acidosis Using Gold-Coated Silicon NanoRoughened Electrode. Anal. Biochem. 2018, 561–562, 1–10.

(27)

Hotani, H.; Miyamoto, H. Dynamic Features of Microtubules as Visualized by Dark-Field Microscopy. Adv. Biophys. 1990, 26, 135–156.

(28)

Cortese, J. D.; Iii, B. S.; Frieden, C.; Elson, E. L. Actin Polymerization Induces a Shape Change in Actin-Containing Vesicles. Proc. Natl. Acad. Sci. 1989, 86 (August), 5773– 5777.

(29)

Galland, R.; Leduc, P.; Guérin, C.; Peyrade, D.; Blanchoin, L.; Théry, M. Fabrication of Three-Dimensional Electrical Connections by Means of Directed Actin Self-Organization. Nat. Mater. 2013, 12 (5), 416–421.

(30)

Staruschenko, A.; Negulyaev, Y. A.; Morachevskaya, E. A. Actin Cytoskeleton Disassembly Affects Conductive Properties of Stretch-Activated Cation Channels in Leukaemia Cells. Biochim. Biophys. Acta - Biomembr. 2005, 1669 (1), 53–60.

(31)

Tuszyński, J. A.; Portet, S.; Dixon, J. M.; Luxford, C.; Cantiello, H. F. Ionic Wave Propagation along Actin Filaments. Biophys. J. 2004, 86 (4), 1890–1903.

(32)

Harner-Foreman, N.; Vadakekolathu, J.; Laversin, S. A.; Mathieu, M. G.; Reeder, S.; Pockley, A. G.; Rees, R. C.; Boocock, D. J. A Novel Spontaneous Model of EpithelialMesenchymal Transition (EMT) Using a Primary Prostate Cancer Derived Cell Line Demonstrating Distinct Stem-like Characteristics. Sci. Rep. 2017, 7 (January), 40633.

(33)

Oh, J. H.; Hur, H.; Lee, J.-Y.; Kim, Y.; Seo, Y.; Kim, M. H. The Mitotic Checkpoint Regulator RAE1 Induces Aggressive Breast Cancer Cell Phenotypes by Mediating Epithelial-Mesenchymal Transition. Sci. Rep. 2017, 7 (February), 42256.

(34)

Etayash, H.; Jiang, K.; Azmi, S.; Thundat, T.; Kaur, K. Real-Time Detection of Breast Cancer Cells Using Peptide-Functionalized Microcantilever Arrays. Sci. Rep. 2015, 5(1), 1–13.

(35)

Shashaani, H.; Faramarzpour, M.; Hassanpour, M.; Namdar, N.; Alikhani, A.; Abdolahad, M. Silicon Nanowire Based Biosensing Platform for Electrochemical Sensing of Mebendazole Drug Activity on Breast Cancer Cells. Biosens. Bioelectron. 2016, 85, 363– 370.

(36)

Yvon, A. C.; Wadsworth, P.; Jordan, M. A. Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells. Mol. Biol. Cell 1999, 10 (4), 947–959.

(37)

Pantziarka, P.; Bouche, G.; Meheus, L.; Sukhatme, V.; Sukhatme, V. P. Repurposing Drugs in Oncology (ReDO)-Mebendazole as an Anti-Cancer Agent. Ecancermedicalscience 2014, 8, 443. 22 ACS Paragon Plus Environment

Page 22 of 26

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

(38)

Mao, Y.; Shin, K.-S.; Wang, X. Semiconductor Electronic Label-Free Assay for Predictive Toxicology. Sci. Rep. 2016, 6 (April), 24982.

(39)

Lee, J.; Jang, J.; Choi, B.; Yoon, J.; Kim, J.-Y.; Choi, Y.-K.; Myong Kim, D.; Hwan Kim, D.; Choi, S.-J. A Highly Responsive Silicon Nanowire/Amplifier MOSFET Hybrid Biosensor. Sci. Rep. 2015, 5, 12286.

(40)

Zhou, W.; Dai, X.; Lieber, C. M. Advances in Nanowire Bioelectronics. Reports Prog. Phys. 2017, 80 (1), 016701.

(41)

Lee, J. H.; Zhang, A.; You, S. S.; Lieber, C. M. Spontaneous Internalization of Cell Penetrating Peptide-Modified Nanowires into Primary Neurons. Nano Lett. 2016, 16 (2), 1509–1513.

(42)

Duan, X.; Fu, T.-M.; Liu, J.; Lieber, C. M. Nanoelectronics-Biology Frontier: From Nanoscopic Probes for Action Potential Recording in Live Cells to Three-Dimensional Cyborg Tissues. Nano Today 2013, 8 (4), 351–373.

(43)

Zhang, A.; Lieber, C. M. Nano-Bioelectronics. Chem. Rev. 2016, 116 (1), 215–257.

(44)

Alikhani, A.; Gharooni, M.; Abiri, H.; Farokhmanesh, F.; Abdolahad, M. Tracing the PH Dependent Activation of Autophagy in Cancer Cells by Silicon Nanowire-Based Impedance Biosensor. J. Pharm. Biomed. Anal. 2018, 154, 158–165.

(45)

Abdolahad, M.; Taghinejad, H.; Saeidi, a.; Taghinejad, M.; Janmaleki, M.; Mohajerzadeh, S. Cell Membrane Electrical Charge Investigations by Silicon Nanowires Incorporated Field Effect Transistor (SiNWFET) Suitable in Cancer Research. RSC Adv. 2014, 4 (15), 7425.

(46)

Elliott, G.; O’Hare, P. Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection. J. Virol. 1999, 73 (5), 4110–4119.

(47)

Hosseini, S. A.; Abdolahad, M.; Zanganeh, S.; Dahmardeh, M.; Gharooni, M.; Abiri, H.; Alikhani, A.; Mohajerzadeh, S.; Mashinchian, O. Nanoelectromechanical Chip (NELMEC) Combination of Nanoelectronics and Microfluidics to Diagnose Epithelial and Mesenchymal Circulating Tumor Cells from Leukocytes. Small 2016, 12 (7), 883– 891.

(48)

Choi, S. G.; Lee, S.-E.; Kang, B.-S.; Ng, C. L.; Davaa, E.; Park, J.-S. Thermosensitive and Mucoadhesive Sol-Gel Composites of Paclitaxel/Dimethyl-β-Cyclodextrin for Buccal Delivery. PLoS One 2014, 9 (10), e109090.

(49)

Wang, L.; Lu, Æ. Y.; Morris, Æ. D. L. Antitumor Activity of Albendazole against the Human Colorectal Cancer Cell Line HT-29 : In Vitro and in a Xenograft Model of Peritoneal Carcinomatosis. Cancer Chemother Pharmacol. 2005, 55(5):425-32.

(50)

Pantziarka, P.; Bouche, G.; Meheus, L.; Sukhatme, V.; Sukhatme, V. P. Repurposing Drugs in Oncology (ReDO)-Mebendazole as an Anti-Cancer Agent. Ecancermedicalscience 2014, 8, 443.

(51)

Zanganeh, S.; Khosravi, S.; Namdar, N.; Amiri, M. H.; Gharooni, M.; Abdolahad, M. Electrochemical Approach for Monitoring the Effect of Anti Tubulin Drugs on Breast 23 ACS Paragon Plus Environment

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cancer Cells Based on Silicon Nanograss Electrodes. Anal. Chim. Acta 2016, 938, 72–81. (52)

Hayot, C.; Debeir, O.; Ham, P. Van; Damme, M. Van; Kiss, R.; Decaestecker, C. Characterization of the Activities of Actin-Affecting Drugs on Tumor Cell Migration. Toxicol Appl Pharmacol. 2006, 211(1), 30-40.

(53)

Bubb, M. R.; Spector, I.; Beyer, B. B.; Fosen, K. M. Effects of Jasplakinolide on the Kinetics of Actin Polymerization. J. Biol. Chem. 2000, 275 (7), 5163–5170.

(54)

Abiri, H.; Abdolahad, M.; Gharooni, M.; Ali Hosseini, S.; Janmaleki, M.; Azimi, S.; Hosseini, M.; Mohajerzadeh, S. Monitoring the Spreading Stage of Lung Cells by Silicon Nanowire Electrical Cell Impedance Sensor for Cancer Detection Purposes. Biosens. Bioelectron. 2015, 68, 577–585.

(55)

Abdolahad, M.; Shashaani, H.; Janmaleki, M.; Mohajerzadeh, S. Silicon Nanograss Based Impedance Biosensor for Label Free Detection of Rare Metastatic Cells among Primary Cancerous Colon Cells, Suitable for More Accurate Cancer Staging. Biosens. Bioelectron. 2014, 59, 151–159.

(56)

Umnov, M.; Palusinski, O. A.; Deymier, P. A.; Guzman, R.; Hoying, J.; Barnaby, H.; Yang, Y.; Raghavan, S. Experimental Evaluation of Electrical Conductivity of Microtubules. J. Mater. Sci. 2007, 42 (1), 373–378.

(57)

Ailenberg, M.; Silverman, M. Cytochalasin D Disruption of Actin Filaments in 3T3 Cells Produces an Anti-Apoptotic Response by Activating Gelatinase A Extracellularly and Initiating Intracellular Survival Signals. Biochim. Biophys. Acta - Mol. Cell Res. 2003, 1593 (2), 249–258.

(58)

Buchwalow, I. B.; Böcker, W. Immunohistochemistry: Basics and Methods; Springer Berlin Heidelberg: Berlin, Heidelberg, 2010.

(59)

Gharooni, M.; Abdolahad, M. Bioelectrical Impedimetric Sensor for Single Cell Analysis Based on Nanoroughened Quartz Substrate; Suitable for Cancer Therapeutic Purposes. J. Pharm. Biomed. Anal. 2017, 142, 315–323.

(60)

Badley, R. A.; Woods, A.; Carruthers, L.; Rees, D. A. Cytoskeleton Changes in Fibroblast Adhesion and Detachment. J. Cell Sci. 1980, 43, 379–390.

(61)

Abdolahad, M.; Janmaleki, M.; Taghinejad, M.; Taghnejad, H.; Salehi, F.; Mohajerzadeh, S. Single-Cell Resolution Diagnosis of Cancer Cells by Carbon Nanotube Electrical Spectroscopy. Nanoscale 2013, 5 (8), 3421.

(62)

Abdolahad, M.; Taghinejad, M.; Taghinejad, H.; Mashinchian, O.; Janmaleki, M.; Mohajerzadeh, S.; Mahmoudi, M.; Saeidi, A.; Azimi, S. Single-Cell Correlative Nanoelectromechanosensing Approach to Detect Cancerous Transformation: Monitoring the Function of F-Actin Microfilaments in the Modulation of Ion Channel Activity. Nanoscale 2015, 7 (5), 1879–1887.

(63)

Moghtaderi, H.; Sepehri, H.; Delphi, L.; Attari, F. Gallic Acid and Curcumin Induce Cytotoxicity and Apoptosis in Human Breast Cancer Cell MDA-MB-231. Bioimpacts 2018, 8 (3), 185–194. 24 ACS Paragon Plus Environment

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(64)

Moghtaderi, H.; Sepehri, H.; Attari, F. Combination of Arabinogalactan and Curcumin Induces Apoptosis in Breast Cancer Cells in Vitro and Inhibits Tumor Growth via Overexpression of P53 Level in Vivo. Biomed. Pharmacother. 2017, 88, 582–594.

(65)

Nikshoar, M. S.; Khayamian, M. A.; Ansaryan, S.; Sanati, H.; Gharooni, M.; Farahmand, L.; Rezakhanloo, F.; Majidzadeh-A, K.; Hoseinpour, P.; Dadgari, S.; et al. Metas-Chip Precisely Identifies Presence of Micrometastasis in Live Biopsy Samples by Label Free Approach. Nat. Commun. 2017, 8 (1), 2175.


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monitoring the electrical functions of actins and microtubules in drug resistance assay of cancer cells 217x164mm (96 x 96 DPI)

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Bioelectronics of cellular cytoskeleton; Monitoring the conducting

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